U.S. patent application number 14/780043 was filed with the patent office on 2016-03-17 for modified tgf-beta oligonucleotides.
This patent application is currently assigned to Isarna Therapeutics GmbH. The applicant listed for this patent is ISARNA THERAPEUTICS GMBH. Invention is credited to Michel JANICOT, Frank JASCHINSKI, Eugen UHLMANN.
Application Number | 20160076037 14/780043 |
Document ID | / |
Family ID | 50390096 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160076037 |
Kind Code |
A1 |
JASCHINSKI; Frank ; et
al. |
March 17, 2016 |
MODIFIED TGF-BETA OLIGONUCLEOTIDES
Abstract
The invention refers to an oligonucleotide consisting of 10 to
20 nucleotides of selected regions of the TGF-beta1, TGF-beta2 or
TGF-beta3 nucleic acid sequence, which comprises modified
nucleotides such as LNA, ENA, polyalkylene oxide-, 2'-fluoro,
2'-O-methoxy and/or 2'-O-methyl modified nucleotides. The selected
regions are preferably the region of nucleic acid no. 1380 to 1510,
no. 1660 to 1680, no. 2390 to 2410, or no. 2740 to 2810 of the
TGF-beta2 nucleic acid sequence of SEQ ID NO. 1, specific regions
of the TGF-beta1 nucleic acid sequence of SEQ ID NO. 149, or
specific regions of the TGF-beta3 nucleic acid sequence of SEQ ID
No. 267. The invention further relates to pharmaceutical
compositions comprising such oligonucleotide, wherein the
composition or the oligonucleotide is used in the prevention and/or
treatment of a malignant and/or benign tumor, an immunologic
disease, fibrosis, glaucoma, etc.
Inventors: |
JASCHINSKI; Frank;
(Obertraubling, DE) ; JANICOT; Michel; (Brussels,
BE) ; UHLMANN; Eugen; (Glashutten, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISARNA THERAPEUTICS GMBH |
Munchen |
|
DE |
|
|
Assignee: |
Isarna Therapeutics GmbH
|
Family ID: |
50390096 |
Appl. No.: |
14/780043 |
Filed: |
March 27, 2014 |
PCT Filed: |
March 27, 2014 |
PCT NO: |
PCT/EP2014/056221 |
371 Date: |
September 25, 2015 |
Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
A61P 25/16 20180101;
C12N 2310/351 20130101; A61P 17/06 20180101; C12N 2310/11 20130101;
A61P 35/00 20180101; C12N 2310/341 20130101; A61P 17/00 20180101;
A61P 35/02 20180101; C12N 15/1136 20130101; A61P 25/28 20180101;
A61P 1/16 20180101; C12N 2310/3231 20130101; C12N 2310/321
20130101; C12N 2310/3525 20130101; C12N 2310/321 20130101; C12N
2310/3521 20130101; C12N 2310/322 20130101; C12N 2310/3533
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2013 |
EP |
13161474.5 |
Jun 20, 2013 |
EP |
13173078.0 |
Dec 30, 2013 |
EP |
13199826.2 |
Claims
1. An oligonucleotide consisting of 12 to 18 nucleotides of the
TGF-beta2 nucleic acid sequence of SEQ ID NO. 1, wherein one or
more nucleotide(s) of the oligonucleotide is/are a LNA modified,
and wherein the modified nucleotide is a LNA, and/or an ENA,
polyalkylene oxide-, 2'-fluoro-, 2'-O-methoxy-, and/or
2'0-methyl-modified nucleotide.
2. The oligonucleotide according to claim 1, said oligonucleotide
comprising 12 to 18 nucleotides of the region of nucleic acid no.
1380 to 1510, no. 1660 to 1680, no. 2390 to 2410, or no. 2740 to
2810 of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 1.
3. The oligonucleotide according to claim 1, wherein the modified
nucleotide is located at the 5'- and/or 3'-end of the
oligonucleotide.
4. The oligonucleotide according to claim 1, wherein the
oligonucleotide comprises a sequence selected from the group
consisting of SEQ ID NO. 46, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO.
4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID
NO. 9, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,
SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID
NO. 25, SEQ ID NO. 31, SEQ ID NO. 35, SEQ ID NO. 44, SEQ ID NO. 47,
SEQ ID NO. 57, SEQ ID NO. 61, SEQ ID NO. 63, SEQ ID NO. 73, SEQ ID
NO. 95, and SEQ ID NO. 103.
5. The oligonucleotide according to claim 1, wherein the
oligonucleotide is selected from the group consisting of
CAAAGTATTTGGTCTCC (ASPH47), ACCTCCTTGGCGTAGTA (ASPH01),
ACCTCCTTGGCGTAGTA (ASPH02), CCTCCTTGGCGTAGTA (ASPH03),
CCTCCTTGGCGTAGTA (ASPH04), CTCCTTGGCGTAGTA (ASPH05),
CTCCTTGGCGTAGTA (ASPH06), CTCCTTGGCGTAGTA (ASPH07), TCCTTGGCGTAGTA
(ASPH08), C AGAAGTTGG CAT (ASPH09), C AGAAGTTGG CAT (ASPH10),
CTGCCCGCGGAT (ASPH15), TCTGCCCGCGGAT (ASPH17), TCGCGCTCGCAGGC
(ASPH22), GGATCTGCCCGCGGA (ASPH26), GGATCTGCCCGCGGA (ASPH27), C GAT
C CTCTTG CG C AT (ASPH30), GGCGGGATGGCAT (ASPH35), GACCAGATGCAGGA
(ASPH36), CTTGCTCAGGATCTGCC (ASPH37), TCTGTAGGAGGGC (ASPH45),
CCTTAAGCCATCCATGA (ASPH48), TCTGAACTAGTACCGCC (ASPH65),
TACTATTATGGCATCCC (ASPH69), AG C GTAATTGGTC AT C A (ASPH71),
GCGACCGTGACCAGAT (ASPH80), AACTAGTACCGCCTTT (ASPH82),
GCGCGACCGTGACC (ASPH98), ACCACTAGAGCACC (ASPH105), AGCGCGACCGTGA
(ASPH111), GGATCGCCTCGAT (ASPH112), CTAGTACCGCCTT (ASPH115),
CCGCGGATCGCC (ASPH119), GACCGTGACCAGAT (ASPH121), GACCGTGACCAGAT
(ASPH153).
6. A pharmaceutical composition comprising the oligonucleotide
according to claim 1 and a pharmaceutically acceptable carrier.
7. A method of preventing and/or treating a malignant and/or benign
tumor, fibrosis, cirrhosis, scleroderma or related dermatologic
diseases, or a CNS disease, comprising the oligonucleotide
according to claim 1.
8. The method according to claim 7, wherein the method is directed
to preventing and/or treating a malignant and/or benign tumor, and
wherein the tumor is at least one of solid tumors, blood born
tumors, leukemias, tumor metastasis, hemangiomas, acoustic
neuromas, neurofibromas, trachomas, pyogenic granulomas, psoriasis,
astrocytoma, acoustic neuroma, blastoma, Ewing's tumor,
craniopharyngioma, ependymoma, medulloblastoma, glioma,
hemangloblastoma, Hodgkins-lymphoma, medullablastoma, leukaemia,
mesothelioma, neuroblastoma, neurofibroma, non-Hodgkins lymphoma,
pinealoma, retinoblastoma, sarcoma, seminoma, trachomas, Wilm's
tumor, bile duct carcinoma, bladder carcinoma, brain tumor, breast
cancer, bronchogenic carcinoma, carcinoma of the kidney, cervical
cancer, choriocarcinoma, choroidcarcinoma, cystadenocarcinome,
embryonal carcinoma, epithelial carcinoma, esophageal cancer,
cervical carcinoma, colon carcinoma, colorectal carcinoma,
endometrial cancer, gallbladder cancer, gastric cancer, head
cancer, liver carcinoma, lung carcinoma, medullary carcinoma, neck
cancer, non-small-cell bronchogenic/lung carcinoma, ovarian cancer,
pancreas carcinoma, papillary carcinoma, papillary adenocarcinoma,
prostata cancer, small intestine carcinoma, prostate carcinoma,
rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer,
small-cell bronchogenic/lung carcinoma, squamous cell carcinoma,
sebaceous gland carcinoma, testicular carcinoma, and uterine
cancer
9. A method of preventing and/or treating a malignant and/or benign
tumor, fibrosis, cirrhosis, scleroderma or related dermatologic
diseases, or a CNS disease, comprising the pharmaceutical
composition according to claim 6.
10. The method according to claim 9, wherein the method is directed
to preventing and/or treating a malignant and/or benign tumor, and
wherein the tumor is at least one of solid tumors, blood born
tumors, leukemias, tumor metastasis, hemangiomas, acoustic
neuromas, neurofibromas, trachomas, pyogenic granulomas, psoriasis,
astrocytoma, acoustic neuroma, blastoma, Ewing's tumor,
craniopharyngioma, ependymoma, medulloblastoma, glioma,
hemangloblastoma, Hodgkins-lymphoma, medullablastoma, leukaemia,
mesothelioma, neuroblastoma, neurofibroma, non-Hodgkins lymphoma,
pinealoma, retinoblastoma, sarcoma, seminoma, trachomas, Wilm's
tumor, bile duct carcinoma, bladder carcinoma, brain tumor, breast
cancer, bronchogenic carcinoma, carcinoma of the kidney, cervical
cancer, choriocarcinoma, choroidcarcinoma, cystadenocarcinome,
embryonal carcinoma, epithelial carcinoma, esophageal cancer,
cervical carcinoma, colon carcinoma, colorectal carcinoma,
endometrial cancer, gallbladder cancer, gastric cancer, head
cancer, liver carcinoma, lung carcinoma, medullary carcinoma, neck
cancer, non-small-cell bronchogenic/lung carcinoma, ovarian cancer,
pancreas carcinoma, papillary carcinoma, papillary adenocarcinoma,
prostata cancer, small intestine carcinoma, prostate carcinoma,
rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer,
small-cell bronchogenic/lung carcinoma, squamous cell carcinoma,
sebaceous gland carcinoma, testicular carcinoma, and uterine cancer
Description
[0001] The invention is directed to oligonucleotides consisting of
10 to 20 nucleotides of elected regions of the TGF-beta2 nucleic
acid sequence, alternatively elected of the TGF-beta1 or TGF-beta3
nucleic acid sequence, which comprise modified nucleotides such as
LNA, ENA, polyalkylene oxide-, 2'-fluoro, 2'-O-methoxy and/or
2'-O-methyl modified nucleotides.
TECHNICAL BACKGROUND
[0002] Transforming growth factor beta (TGF-beta) is a protein that
controls proliferation, cellular differentiation, and other
functions in most cells. It is a type of cytokine which plays
amongst others a role in immunity, cancer, heart disease, diabetes,
Marfan syndrome, Loeys-Dietz syndrome, Parkinson's disease, and
AIDS.
[0003] TGF-beta is a secreted protein that exists in at least three
isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) encoded by different
genes but sharing strong sequence and structure homologies.
TGF-beta acts as an antiproliferative factor in normal epithelial
cells and at early stages of oncogenesis. However, later in tumor
development TGF-beta can become tumor promoting through mechanisms
including the induction of epithelial-to-mesenchymal transition
(EMT), a process that is thought to contribute to tumor
progression, invasion and metastasis (see "Glycoproteomic analysis
of two mouse mammary cell lines during transforming growth factor
(TGF)-beta induced epithelial to mesenchymal transition" 7.sup.th
space.com.2009-01-08. Retrieved: 2009 Jan. 29).
[0004] In normal (epithelial) cells, TGF-beta stops the cell cycle
at the G1 stage (and stops cell proliferation), induce
differentiation, or promote apoptosis. When a cell is transformed
into a cancer cell, TGF-beta no longer suppresses cell
proliferation, which is often the result of mutations in the
signaling pathway, and cancer cells proliferate.
[0005] Proliferation of stromal fibroblasts is also induced by
TGF-beta. Both cells increase their production of TGF-beta. This
TGF-beta acts on the surrounding stromal cells, immune cells,
endothelial, smooth-muscle cells, and tumor microenvironment (see
Pickup et al., "The roles of TGF.beta. in the tumour
microenvironment", Nature Reviews Cancer (2013), 13: 788-799).
Thereby, it promotes angiogenesis, and by suppressing proliferation
and activation of immune cells it causes immunosuppression.
[0006] TGF-beta1-deficient mice die from cardiac, pulmonary, and
gastric inflammation, suggesting that TGF-beta has a vital role in
suppressing the activation and proliferation of inflammatory cells.
Smad3 is one of the key elements in TGF-beta dependent downstream
signaling pathways. Smad3-deficient mice develop chronic mucosal
infections due to impairment of T-cell activation and mucosal
immunity, suggesting a key role for TGF-beta in these processes.
With respect to cancer, the production and secretion of TGF-beta by
certain cancer cells suppress the activities of infiltrating immune
cells, thereby helping the tumor to escape host immunosurveillance.
This immunosuppressive effect may be another important mechanism by
which TGF-beta stimulates the growth of late-stage tumors (see
Blobe G C et al., May 2000, "Role of transforming growth factor
beta in human disease", N. Engl. J. Med. 342 (18), 1350-1358).
TGF-beta also converts effector T-cells, which normally attack
cancer with an inflammatory (immune) reaction, into regulatory
(suppressor) T-cells, which turn off the inflammatory reaction.
[0007] Further, TGF-beta is one of the most potent regulators of
the production and deposition of extracellular matrix. It
stimulates the production and affects the adhesive properties of
the extracellular matrix by two major mechanisms. First, TGF-beta
stimulates fibroblasts and other cells to produce
extracellular-matrix proteins and cell-adhesion proteins, including
collagen, fibronectin, and integrins. Second, TGF-beta decreases
the production of enzymes that degrade the extracellular matrix,
including collagenase, heparinase, and stromelysin, and increases
the production of proteins that inhibit enzymes that degrade the
extracellular matrix, including plasminogen-activator inhibitor
type 1 and tissue inhibitor of metalloprotease. The net effect of
these changes is to increase the production of extracellular-matrix
proteins and either to increase or to decrease the adhesive
properties of cells in a cell-specific manner. In many cancer cells
the production of TGF-beta is increased, which increases the
invasiveness of the cells by increasing their proteolytic activity
and promoting their binding to cell-adhesion molecules (see Blobe G
C et al., May 2000, "Role of transforming growth factor beta in
human disease", N. Engl. J. Med. 342 (18), 1350-1358).
[0008] Thus, therapeutic agents which are able to influence
TGF-beta expression and activity, respectively, are essential in
particular for use in preventing and/or treating TGF-beta linked
diseases. EP 1008649 and EP 0695354, for example, disclose
oligonucleotides hybridizing with the mRNA of TGF-beta1 and/or
TGF-beta2, and which are suitable to be used for manufacturing
pharmaceutical compositions for example for preventing and/or
treating cancer. None of these oligonucleotides comprises
modifications such as LNA, ENA etc.
[0009] WO 2003/85110, WO 2005/061710, and WO 2008/138904 for
example refer to oligonucleotides comprising modifications of the
nucleotides, which are directed to the inhibition of HIF-1A, Bcl-2
and HER3, respectively, usable in the treatment of cancer.
[0010] Criteria for the selection of oligonucleotides are mainly
the length of the oligonucleotide, the GC-percentage, the tendency
for hairpin formation, dimerization and the melting temperature
(Tm). In general, high Tm (melting temperature) is preferred.
Furthermore, the oligonucleotides must be specific for the target
mRNA and shall not hybridize to non-target mRNAs in order to
decrease potential off-target effects.
[0011] Hence, there is a high scientific and medical need for
therapeutic agents, which reduce or inhibit TGF-beta expression
and/or activity. Particularly, there is a long-standing need for
oligonucleotides such as antisense oligonucleotides, which
specifically interact and thus, reduce or inhibit the expression of
TGF-beta1, TGF-beta2, and/or TGF-beta3, as well as
oligonucleotides, which specifically inhibit TGF-beta1 and
TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3,
without causing any (severe) side effects.
SUMMARY OF THE INVENTION
[0012] The present invention refers to oligonucleotides consisting
of 10 to 20, preferably 12 to 18 nucleotides of the TGF-beta2
nucleic acid sequence of SEQ ID NO. 1 (see FIG. 2), or of the
TGF-beta1 nucleic acid sequence of SEQ ID NO. 335 (see FIG. 12), or
of the TGF-beta3 nucleic acid sequence of SEQ ID NO. 336 (see FIG.
25), wherein one or more nucleotide(s) of the oligonucleotide
is/are modified. Some of the oligonucleotides of the present
invention correspond to TGF-beta1, TGF-beta2, and TGF-beta3, or to
TGF-beta1 and TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2
and TGF-beta3. Preferred oligonucleotides comprise or consist of
one of SEQ ID NO. 2 to 149 (TGF-beta2), of one of SEQ ID No.
150-334 (TGF-beta1), or of one of SEQ ID No. 337-402 (TGF-beta3),
which are presented in Table 1.
[0013] In particular, oligonucleotides of the present invention
comprise or consist of 10 to 20, more preferred of 12 to 18
nucleotides of the region of nucleic acid no. 1380 to 1510 of SEQ
ID NO. 1, wherein one or more nucleotide(s) of the oligonucleotide
is/are modified. These oligonucleotides are highly effective in the
reduction and inhibition of TGF-beta2 expression and activity,
respectively. A preferred oligonucleotide comprises or consists of
SEQ ID NO. 2 (e.g., ASPH36: GACCAGATGCAGGA), SEQ ID NO. 3 (e.g.,
ASPH80: GCGACCGTGACCAGAT), SEQ ID NO. 4 (e.g., ASPH98:
GCGCGACCGTGACC), SEQ ID NO. 5 (e.g., ASPH111: AGCGCGACCGTGA), or
SEQ ID NO. 6 (e.g., ASPH121 or ASPH153: GACCGTGACCAGAT), SEQ ID NO.
7 (e.g., ASPH15: CTGCCCGCGGAT), SEQ ID NO. 8 (e.g., ASPH17:
TCTGCCCGCGGAT), SEQ ID NO. 9 (e.g., ASPH26 or ASPH27:
GGATCTGCCCGCGGA), SEQ ID NO. 10 (e.g., ASPH37: CTTGCTCAGGATCTGCC),
SEQ ID NO. 11 (e.g., ASPH52 or 53: GCTCAGGATCTGCCCGCGGA), SEQ ID
NO. 12 (e.g., ASPH112: GGATCGCCTCGAT), SEQ ID NO. 13 (e.g.,
ASPH119: CCGCGGATCGCC), or SEQ ID NO. 31 (e.g., ASPH30:
CGATCCTCTTGCGCAT).
[0014] In another embodiment the invention refers to an
oligonucleotide, comprising or consisting of 10 to 20, more
preferred of 12 to 18 nucleotides of the region of nucleic acid no.
2740 to 2810 of the TGF-beta2 nucleic acid sequence of SEQ ID NO.
1, wherein one or more nucleotide(s) of the oligonucleotide is/are
modified. These oligonucleotides are highly effective in the
reduction and inhibition of TGF-beta2 expression and activity,
respectively. A preferred oligonucleotide comprises or consists of
SEQ ID NO. 57 (e.g., ASPH65: TCTGAACTAGTACCGCC), SEQ ID NO. 73
(e.g., ASPH82: AACTAGTACCGCCTTT), or SEQ ID NO. 103 (e.g., ASPH115:
CTAGTACCGCCTT).
[0015] In a further embodiment the invention refers to an
oligonucleotide, comprising or consisting of 10 to 20, more
preferred of 12 to 18 nucleotides of the region of nucleic acid no.
1660 to 1680 of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 1
wherein one or more nucleotide(s) of the oligonucleotide is/are
modified. These oligonucleotides are highly effective in the
reduction and inhibition of TGF-beta1 and/or TGF-beta2 expression
and activity, respectively. A preferred oligonucleotide comprises
or consists of SEQ ID NO. 14 (e.g., ASHP01 or ASPH02:
ACCTCCTTGGCGTAGTA), SEQ ID NO. 15 (e.g., ASPH03 or ASPH04:
CCTCCTTGGCGTAGTA), SEQ ID NO. 16 (e.g., ASPH05, ASPH06, or ASPH07:
CTCCTTGGCGTAGTA), or SEQ ID NO.17 (e.g., ASPH08:
TCCTTGGCGTAGTA).
[0016] In another embodiment the invention relates to an
oligonucleotide, comprising or consisting of 10 to 20, more
preferred of 12 to 18 nucleotides, most preferably 13 nucleotides
of the region of nucleic acid no. 2390 to 2410 of the TGF-beta2
nucleic acid sequence of SEQ ID NO. 1 wherein one or more
nucleotide(s) of the oligonucleotide is/are modified. These
oligonucleotides are highly effective in the reduction and
inhibition of TGF-beta1, TGF-beta2, and/or TGF-beta3 expression and
activity, respectively. A preferred oligonucleotide comprises or
consists of SEQ ID NO. 18 (e.g., ASPH9 or ASPH10:
CAGAAGTTGGCAT).
[0017] In another embodiment the invention relates to an
oligonucleotide, comprising or consisting of 10 to 20, more
preferred of 12 to 18 nucleotides of the TGF-beta2 nucleic acid
sequence of SEQ ID NO. 1 wherein one or more nucleotide(s) of the
oligonucleotide is/are modified. These oligonucleotides are highly
effective in the reduction and inhibition of TGF-beta1, TGF-beta2,
and/or TGF-beta3, most preferably of TGF-beta2 expression and
activity, respectively. A preferred oligonucleotide comprises or
consists of one of SEQ ID NO. 19 to 56, 58 to 72, 74 to 102, 104 to
138 (e.g., ASHP11-ASPH14, ASPH16, ASPH18-ASPH25, ASPH28-ASPH35,
ASPH38-ASPH51, ASPH60-64, ASPH66-ASPH79, ASPH81, ASPH83-ASPH97,
ASPH99-ASPH110, ASPH113, ASPH114, ASPH116-118, ASPH120,
ASPH122-ASPH152, ASPH154-ASPH183, or T-LNA (SEQ ID NO: 144)).
[0018] Preferred oligonucleotides of the present invention are
ASPH01, ASPH03, ASPH05, ASPH17, ASPH22, ASPH26, ASPH27, ASPH35,
ASPH36, ASPH37, ASPH45, ASPH47, ASPH48, ASPH65, ASPH69, ASPH71,
ASPH80, ASPH82, ASPH98, ASPH105, ASPH115, ASPH190, ASPH191,
ASPH192, and ASPH193, respectively.
[0019] Further preferred oligonucleotides of the present invention
are ASPH1000 to ASPH1132 as shown in Table 1, which preferably
inhibit the expression and/or activity of TGFbeta1 mRNA. Preferred
oligonucleotides this group are for example ASPH1047, ASPH1051,
ASPH1059, ASPH1106, ASPH1139, ASPH1150, ASPH1162, ASPH1163,
ASPH1175, ASPH1178, and ASPH1181, respectively.
[0020] In an alternative embodiment oligonucleotides are preferably
inhibiting the expression and/or activity of TGF-beta3 mRNA. Such
oligonucleotides are for example ASPH2000, ASPH2001, ASPH2002,
ASPH2003, ASPH2004, ASPH2005, ASPH2006, ASPH2007, ASPH2008,
ASPH2009, ASPH2010, ASPH2011, ASPH2012, ASPH2013, ASPH2014,
ASPH2015, ASPH2016, ASPH2017, ASPH2018, ASPH2019, ASPH2020,
ASPH2021, ASPH2022, ASPH2023, ASPH2024, ASPH2025, ASPH2026,
ASPH2027, ASPH2028, ASPH2029, ASPH2030, ASPH2031, ASPH2032,
ASPH2033, ASPH2034, ASPH2035, ASPH2036, ASPH2037, ASPH2038,
ASPH2039, ASPH2040, ASPH2041, ASPH2042, ASPH2043, ASPH2044,
ASPH2045, ASPH2046, ASPH2047, ASPH2048, ASPH2049, ASPH2050,
ASPH2051, ASPH2052, ASPH2053, ASPH2054, ASPH2055, ASPH2056,
ASPH2057, ASPH2058, ASPH2059, ASPH2060, ASPH2061, ASPH2062,
ASPH2063, ASPH2064, ASPH2065, and ASPH2066, respectively.
[0021] Oligonucleotides of the present invention show an unexpected
strong and specific inhibition of TGF-beta1, TGF-beta2, or
TGF-beta3, or TGF-beta1 and TGF-beta2.
[0022] Alternatively, oligonucleotides of the present invention
show strong and specific inhibition of TGF-beta1 and TGF-beta3, or
TGF-beta1 and TGF-beta2, or TGF-beta2 and TGF-beta3, and in a
further alternative TGF-beta1, TGF-beta2 and TGF-beta3.
[0023] Modifications of one or more nucleotides of the
oligonucleotides of the present invention are selected from the
group consisting of LNA, ENA, polyalkylene oxide such as
triethylene glycol (TEG), 2'-fluoro, 2'-O-methoxy and 2'-O-methyl.
The modifications are preferably located at the 5'- and/or 3'-end
of the oligonucleotide. An oligonucleotide comprising such modified
nucleotide is a modified oligonucleotide.
[0024] Modified nucleotides are for example arranged in a row, one
directly next to the other, or in different patterns, where one or
more unmodified nucleotides follow a modified nucleotide. For
example an oligonucleotide starts with one or more modified
nucleotides followed by one or more, e.g., one, two, three or four,
unmodified or unlocked nucleotides followed again by one or more
modified nucleotides. In one embodiment both ends of the
oligonucleotide comprise an identical pattern of modified and
unmodified or unlocked nucleotides. In another embodiment, the
pattern of modifications at the 3'- and 5'-end differ including
that one end does not comprise a modified nucleotide. Preferably
the modified oligonucleotides comprise a series of 8 or 9 unlocked
nucleotides.
[0025] Alternatively, a nucleotide at any other position in the
oligonucleotide is modified, or at least one nucleotide at the 5'-
and/or 3'-end of the oligonucleotide and at any other position in
the oligonucleotide. For example ASPH1071, ASPH1100, ASPH1109, ASPH
1110, ASPH1111, ASPH1115, ASPH1126, ASPH1127 and ASPH1128 belong to
a group of TGF-beta oligonucleotides, for example TGF-beta1
oligonucleotides, which comprises modified nucleosides such as LNA,
ENA etc. in different patterns, e.g., separated from each other by
an unlocked nucleotide. The oligonucleotides comprise either one
type of modification, or one or more different modifications.
Optionally, at least one phosphate linkage between two consecutive
nucleotides (modified or unmodified) of the oligonucleotide is a
phosphorothioate or a methylphosphonate. In a preferred embodiment,
the oligonucleotides of the present invention are
phosphorothioates.
[0026] Moreover, the present invention refers to TGF-beta antisense
oligonucleotides, which interact and inhibit the expression of more
than one TGF-beta isoform, even if the oligonucleotide is not 100%
complementary to the TGF-beta1, TGF-beta2 and/or TGF-beta3
sequence. Such antisense oligonucleotides are for example ASPH1024,
ASPH1096, ASPH1131 and ASPH1132, respectively. These
oligonucleotides preferably interact with TGF-beta sequences of
different species such as human and mouse as for example ASPH1131
and ASPH1132, respectively.
[0027] All the oligonucleotides of the different embodiments are
for use in a method of the prevention and/or treatment of a
malignant or a benign tumor, an immunologic disease, fibrosis
(e.g., idiopathic pulmonary fibrosis, renal fibrosis, kidney
fibrosis), cirrhosis (e.g., liver cirrhosis), scleroderma or
related dermatologic diseases, an eye disease such as glaucoma or
posterior capsular opacification (PCO), a CNS disease, hair loss
etc.
FIGURES
[0028] FIG. 1 presents examples of nucleotide modifications.
[0029] FIG. 2 shows the nucleic acid sequence of human TGF-beta2
mRNA (NM.sub.--003238.3).
[0030] FIGS. 3a) to 3c) depict the inhibition of the expression of
TGF-beta1 and TGF-beta2 mRNA in human A172 glioma cells. A172 cells
were transfected with different modified oligonucleotides in a dose
of 10 nM (in the presence of a transfecting agent), and the
inhibition of the TGF-beta1 (white columns) and TGF-beta2 (black
columns) mRNA expression was measured 24 h after transfection. FIG.
3a) refers to the results for the modified oligonucleotides ASPH01,
ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08, ASPH09,
ASPH10, ASPH11, ASPH12, ASPH13, ASPH14, ASPH15, ASPH16, ASPH17,
ASPH18, ASPH19, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25, ASPH26,
ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH34, ASPH35,
ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42, ASPH43,
ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50, ASPH51,
ASPH52, ASPH53, and ASPH54; FIG. 3b) to the results for the
modified oligonucleotides ASPH36, ASPH60, ASPH61, ASPH62, ASPH63,
ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70, ASPH71,
ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78, ASPH79,
ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86, ASPH87,
ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94, ASPH95,
ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103,
ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110,
ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117,
ASPH118, and ASPH119; and FIG. 3c) to the results for the modified
oligonucleotides ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122,
ASPH123, ASPH124, ASPH125, ASPH126, ASPH127, ASPH128, ASPH129,
ASPH130, ASPH131, ASPH132, ASPH133, ASPH134, ASPH135, ASPH136,
ASPH137, ASPH138, ASPH139, ASPH140, ASPH141, ASPH142, ASPH143,
ASPH145, ASPH146, ASPH147, ASPH148, ASPH149, ASPH150, ASPH151,
ASPH152, ASPH153, ASPH154, ASPH155, ASPH157, ASPH158, ASPH160,
ASPH161, ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167,
ASPH168, ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174,
ASPH175, ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181,
ASPH182, and ASPH183. Experiments are described in Example 1.
[0031] FIGS. 4a) to 4c) depict the inhibition of the expression of
TGF-beta1 and TGF-beta2 mRNA in human Panc-1 pancreatic cancer
cells. Panc-1 cells were transfected with different modified
oligonucleotides in a dose of 10 nM (in the presence of a
transfecting agent), and the inhibition of the TGF-beta1 (white
columns) and TGF-beta2 (black columns) mRNA expression was measured
24 h after transfection. FIG. 4a) refers to the results for the
modified oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05,
ASPH06, ASPH07, ASPH08, ASPH12, ASPH14, ASPH17, ASPH18, ASPH20,
ASPH21, ASPH22, ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30,
ASPH31, ASPH32, ASPH33, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39,
ASPH40, ASPH41, ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47,
ASPH48, ASPH49, ASPH50, ASPH51, and ASPH52; FIG. 4b) to the results
for the modified oligonucleotides ASPH36, ASPH60, ASPH61, ASPH62,
ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70,
ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78,
ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86,
ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94,
ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103,
ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110,
ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117,
ASPH118, and ASPH119; and FIG. 4c) to the results for the modified
oligonucleotides ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122,
ASPH127, ASPH128, ASPH129, ASPH130, ASPH131, ASPH132, ASPH133,
ASPH135, ASPH136, ASPH137, ASPH139, ASPH141, ASPH142, ASPH143,
ASPH145, ASPH146, ASPH147, ASPH149, ASPH150, ASPH151, ASPH152,
ASPH153, ASPH154, ASPH155, ASPH157, ASPH160, ASPH161, ASPH162,
ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168, ASPH169,
ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175, ASPH176,
ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and ASPH183.
Experiments are described in Example 2.
[0032] FIG. 5 shows the inhibition of the expression of TGF-beta1
and TGF-beta2 mRNA in Panc-1 cells. Panc-1 cells were treated with
different modified oligonucleotides in a dose of 3.3 .mu.M in the
absence of any transfection reagent (gymnotic transfection or
unassisted transfection or gymnotic delivery), and the inhibition
of the TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA
expression was measured after 72 h. FIG. 5 presents the results for
the modified oligonucleotides ASPH17, ASPH18, ASPH22, ASPH25,
ASPH33, ASPH35, ASPH36, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47,
ASPH48, ASPH49, ASPH65, ASPH66, ASPH67, ASPH69, ASPH71, ASPH79,
ASPH80, ASPH82, ASPH88, ASPH89, ASPH90, ASPH91, ASPH98, ASPH99,
ASPH102, ASPH105, ASPH111, ASPH115, ASPH119, ASPH121, ASPH139,
ASPH140, ASPH146, ASPH151, ASPH153, ASPH165, ASPH171, ASPH172,
ASPH176, ASPH178, ASPH180, and ASPH183. Experiments are described
in Example 4.
[0033] FIG. 6 and FIG. 7 present the inhibition of the expression
of TGF-beta1 (FIG. 6a) and TGF-beta2 (FIG. 6b) mRNA as well as the
inhibition of TGF-beta1 (FIG. 7a) and TGF-beta2 (FIG. 7b) protein
in Panc-1 cells. Panc-1 cells were treated with different modified
oligonucleotides in a dose of 10 .mu.M via gymnotic delivery, i.e.,
in the absence of any transfecting reagent, and the inhibition of
the TGF-beta1 and TGF-beta2 mRNA expression and protein was
measured 4 days after transfection. FIG. 6a) and FIG. 6b) show the
results for the modified oligonucleotides ASPH01, ASPH03, ASPH05,
ASPH09, ASPH17, ASPH18, ASPH22, ASPH35, ASPH36, ASPH37, ASPH41,
ASPH45, ASPH46, ASPH47, and ASPH48 on mRNA (FIG. 7a) and protein
(FIG. 7b) level. Experiments are described in Example 5.
[0034] FIG. 8 depicts the dose-dependent effect of modified
oligonucleotides ASPH05 and ASPH36 on TGF-beta1 and TGF-beta2 mRNA
expression. Panc-1 cells were treated for 4 days with 15 .mu.M, 10
.mu.M, 7.5 .mu.M, 5 .mu.M, 2.5 .mu.M, 1.25 .mu.M, or 0.625 .mu.M of
either ASPH05 (dual TGF-beta1 and TGF-beta2 oligonucleotide) or
ASPH36 (selective TGF-beta2 oligonucleotide) modified
oligonucleotide in the absence of a transfection reagent. Remaining
TGF-beta1 (FIG. 8a) or TGF-beta2 mRNA (FIG. 8b) was measured after
4 days. Experiments are described in Example 6.
[0035] FIG. 9 shows the inhibition of the expression of TGF-beta1
and TGF-beta2 mRNA in mouse SMA-560 glioma cells. SMA-560 cells
were transfected with ASPH01, ASPH03, ASPH05, ASPH09, ASPH17,
ASPH18, ASPH22, ASPH26, ASPH36, ASPH37, ASPH41, ASPH42, ASPH45,
ASPH46, ASPH47, or ASPH48 in a dose of 10 nM (in the presence of a
transfecting agent). Inhibition of the mouse TGF-beta1 (white
columns) and TGF-beta2 (black columns) mRNA expression was
determined 24 h after transfection. Experiments are described in
Example 7.
[0036] FIG. 10 presents in vivo data referring to the treatment of
female athymic nude mice with ASPH01, ASPH03, ASPH05, ASPH17,
ASPH22, ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, or ASPH48 at 14
mg/kg body weight by subcutaneous injection for 5 consecutive days.
24 h after the last treatment, mice were sacrificed and mouse
TGF-beta 2 mRNA was quantified in kidney tissue lysates.
Data--representing TGF-beta2 to GAPDH mRNA ratio--are shown as a
box plot in which median values and min. and max. values are
presented (data expressed as n=4, except ASPH46 group n=3).
Experiments are described in Example 8.
[0037] FIG. 11 shows the inhibition of the expression of TGF-beta3
mRNA in Panc-1 cells. Panc-1 cells were treated with ASPH09 in a
dose of 10 .mu.M in the absence of any transfection reagent
(gymnotic transfection or unassisted transfection), and inhibition
of the TGF-beta3 mRNA expression was measured after 4 days. ASPH09
is a pan-specific oligonucleotide inhibiting the expression of
TGF-beta3 as well as TGF-beta1 and TGF-beta2 (FIGS. 6a and 6b).
Experiment is described in Example 9.
[0038] FIG. 12 presents the nucleic acid sequence of human
TGF-beta1 mRNA (NM.sub.--000660.4).
[0039] FIG. 13 depicts the inhibition of the expression of
TGF-beta1 mRNA in human Panc-1 pancreatic cancer cells. Panc-1
cells were transfected with different modified oligonucleotides in
a dose of 10 nM (in the presence of a transfecting agent), and
inhibition of the TGF-beta1 mRNA expression was measured 24 h after
transfection. FIG. 13 refers to the results for the modified
oligonucleotides ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002,
ASPH1003, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008,
ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014,
ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020,
ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027,
ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033,
ASPH1034, ASPH1035, ASPH1036, ASPH1038, ASPH1039, ASPH1040,
ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046,
ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052,
ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059,
ASPH1060, and ASPH1061. Experiments are described in Example
12.
[0040] FIG. 14 shows the inhibition of the expression of TGF-beta1
mRNA in mouse SMA-560 glioma cells. Cells were transfected with
different modified oligonucleotides in a dose of 10 nM (in the
presence of a transfecting agent), and inhibition of the TGF-beta1
mRNA expression was measured 24 h after transfection. FIG. 14
refers to the results for the modified oligonucleotides ASPH09,
ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005,
ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011,
ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017,
ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023,
ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030,
ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036,
ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042,
ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048,
ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054,
ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060,
ASPH1061, and ASPH1062. Experiments are described in Example
13.
[0041] FIG. 15 depicts the inhibition of the expression of
TGF-beta1 and TGF-beta2 mRNA in human A172 cells. Cells were
transfected with different modified oligonucleotides in a dose of
10 nM (in the presence of a transfecting agent), and inhibition of
the TGF-beta1 and TGF-beta2 mRNA expression was measured 24 h after
transfection. FIG. 15 refers to the results for the modified
oligonucleotides ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002,
ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009,
ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015,
ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021,
ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028,
ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034,
ASPH1035, ASPH1036, ASPH1038, ASPH1039, ASPH1040, ASPH1041,
ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047,
ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053,
ASPH1054, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060,
ASPH1061, and ASPH1062. Experiments are described in Example
14.
[0042] FIG. 16 shows the inhibition of the expression of TGF-beta1
and TGF-beta2 mRNA in Panc-1 cells. Panc-1 cells were treated with
different modified oligonucleotides in a dose of 3.3 .mu.M in the
absence of any transfection reagent (gymnotic transfection or
unassisted transfection or gymnotic delivery), and inhibition of
the TGF-beta1 (black columns) and TGF-beta2 (white columns) mRNA
expression was measured after 72 h. FIG. 16 refers to the results
for the modified oligonucleotides ASPH05, ASPH09, ASPH1000,
ASPH1001, ASPH1002, ASPH1004, ASPH1006, ASPH1007, ASPH1008,
ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014,
ASPH1015, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021,
ASPH1022, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029,
ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1037,
ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043,
ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1049, ASPH1050,
ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1055, ASPH1056,
ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, and ASPH1062.
Experiments are described in Example 15.
[0043] FIG. 17 depicts the inhibition of the expression of
TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human A172 cells. Cells
were transfected with different modified oligonucleotides in a dose
of 10 nM (in the presence of a transfecting agent), and inhibition
of the TGF-beta1 (black column), TGF-beta2 (white column) and
TGF-beta3 (striped column) mRNA expression was measured 24 h after
transfection. FIG. 17 refers to the results for the modified
oligonucleotides ASPH09, ASPH1047, ASPH1051, ASPH1059, ASPH1063,
ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069,
ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075,
ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081,
ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087,
ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093,
ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100,
ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106,
ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112,
ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118,
ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124,
ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130,
ASPH1131, and ASPH1132. Experiments are described in Example
16.
[0044] FIG. 18a shows the inhibition of the expression of
TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human Panc-1 and RenCa
cells. Cells were transfected with different modified
oligonucleotides in a dose of 3.3 .mu.M in the absence of any
transfection reagent (gymnotic transfection or unassisted
transfection or gymnotic delivery), and inhibition of the TGF-beta1
(black column), TGF-beta2 (white column) and TGF-beta3 (striped
column) mRNA expression was measured 72 h after transfection. FIG.
18a refers to the results for the modified oligonucleotides
ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068,
ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074,
ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080,
ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086,
ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092,
ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099,
ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105,
ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111,
ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117,
ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123,
ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129,
ASPH1130, ASPH1131, and ASPH1132. FIG. 18b presents the inhibiting
effect of these oligonucleotides in RenCa cells.
[0045] FIG. 19 presents a sequence alignment of ASPH1024 and
ASPH1096 with the human sequence of TGF-beta1, TGF-beta2 and
TGF-beta3 mRNAs. Both oligonucleotides are 100% homologous to the
human sequence of TGF-beta1. ASPH1024 has three mismatches with the
human sequence of TGF-beta2 (FIG. 19a) and two mismatches with
human sequence of TGF-beta3 (FIG. 19b). ASPH1096 has one mismatch
with the human sequence of TGF-beta2 (FIG. 19a), and one mismatch
with the human sequence of TGF-beta3 (FIG. 19b). Both
oligonucleotides show inhibition of different human TGF-beta
isoforms (TGF-beta1, TGF-beta2, and TGF-beta3). For example
ASPH1024 inhibits the expression and activity of TGF-beta1 and
TGF-beta2 (see FIG. 16) and ASPH1096 inhibits the expression and
activity of TGF-beta1, TGF-beta2 and TGF-beta3 as depicted in FIG.
17 for example. ASPH009, which is 100% homologous to the human
sequence of TGF-beta1, TGF-beta2, and TGF-beta3 was used as a
control.
[0046] FIG. 20 shows an alignment of ASPH1131 and ASPH1132 with the
human sequences of TGF-beta1, TGF-beta2 and TGF-beta3 mRNAs. Both
oligonucleotides are 100% homologous to the human sequences of
TGF-beta1 and TGF-beta3. Each of ASPH1131 and ASPH1132 has one
mismatch with the human sequence of TGF-beta2. Both
oligonucleotides strongly inhibit the expression of all three human
isoforms as depicted in FIG. 17 for example.
[0047] FIG. 21 depicts an alignment of ASPH1131 and ASPH1132 with
the murine sequences of TGF-beta1, TGF-beta2 and TGF-beta3 mRNAs.
Both oligonucleotides are 100% homologous to the murine sequences
of TGF-beta1 and TGF-beta3. Each of ASPH1131 and ASPH1132 has two
mismatches with the murine sequence of TGF-beta2. While ASPH1131
potently inhibits murine TGF-beta2 and TGF-beta3, ASPH1132 very
potently suppresses all murine TGF-beta isoforms as depicted in
FIG. 18b for example.
[0048] FIG. 22 shows TGF-beta2 mRNA expression in the kidney of
mice bearing subcutaneous human pancreatic carcinoma Panc-1. Mice
were treated with 1, 3, 10, and 30 mg/kg of ASPH47 after indicated
treatment schedules for 5 days: Q1Dx1-d6 (single SC injection,
termination 5 days later), Q1Dx5-d6 (daily SC injection for 5 days,
termination 24 hours later), and Q1Dx5-d10 (daily SC injection for
5 days, termination 5 days later). TGF-beta 2 expression was
detected by bDNA assay and normalized to GAPDH. Data--representing
TGF-beta2 to GAPDH mRNA ratio--are shown as a box plot in which
median values and min. and max. values are presented (data
expressed as n=10, except n=9 for vehicle and 3 mg/kg Q1Dx1 d6
groups).
[0049] FIG. 23 depicts TGF-beta2 mRNA expression in the kidneys of
mice bearing human pancreatic carcinoma Panc-1 tumors. Mice were
treated with subcutaneous injections of various oligonucleotides
for 5 consecutive days using indicated treatment doses: daily
injection of 1, 5, 15 or 50 mg/kg oligonucleotides for five
consecutive days. TGF-beta2 mRNA expression was detected by bDNA
assay and normalized to GAPDH. Data--representing TGF-beta2 to
GAPDH mRNA ratio--are shown as a box plot in which median values
and min. and max. values are presented (data expressed as n=5).
[0050] FIG. 24 presents TGF-beta2 mRNA expression in subcutaneous
human renal cell carcinomas 786-0 tumors. Mice were treated with a
daily injection of 50 mg/kg oligonucleotides for five consecutive
days. TGF-beta2 and GAPDH mRNA expression was detected by bDNA.
Data--representing TGF-beta2 to GAPDH mRNA ratio--are shown as a
box plot in which median values and min. and max. values are
presented (data expressed as n=10, except for ASPH71 group
n=9).
[0051] FIG. 25 shows the nucleic acid sequence of human TGF-beta3
mRNA (NM.sub.--003239.2).
[0052] FIG. 26 depicts the inhibiting effect of oligonucleotides of
the present invention on the expression of TGF-beta1 and TGF-beta2
protein. Panc-1 cells were transfected with 20, 6.67, 2.22, 0.74,
0.25, 0.08 or 0.009 .mu.M of the modified oligonucleotides ASPH47
(FIG. 26a), ASPH1047 (FIG. 26b), ASPH1106 (FIG. 26c), ASPH1132
(FIG. 26d), or ASPH47 in combination with ASPH1047 (FIG. 26e).
Negative control is the scrambled oligonucleotide (scrLNA) of SEQ
ID No. 145 (FIG. 26f) in concentrations of 40, 13.33, 4.44, 1.48,
0.49, 0.16, 0.05, or 0.02 .mu.M. TGF-beta1 (diamonds) and TGF-beta2
protein (squares) levels in cell supernatants were determined by
ELISA.
[0053] FIG. 27 presents the inhibiting effect of oligonucleotides
of the present invention on the expression of TGF-beta1, TGF-beta2,
and TGF-beta3. Panc-1 cells (FIG. 27a) or RenCa cells (FIG. 27b)
were transfected with 3.3 .mu.M of different TGF-beta specific
oligonucleotides in the absence of a transfecting agent. The
expression of TGF-beta1 (black column), TGF-beta2 (white column)
and TGF-beta3 (striped column) mRNA was determined 72 h after
transfection.
[0054] FIG. 28 depicts the inhibiting effect of oligonucleotides of
the present invention on the expression of TGF-beta1, TGF-beta2,
and TGF-beta3. A172 glioma cells were transfected with 10 nM of
different TGF-beta specific oligonucleotides in the presence of
transfecting agent. The expression of TGF-beta1 (black column),
TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was
determined 24 h after transfection.
[0055] FIGS. 29a and 29b present a compared analysis of
time-dependent plasma (29a) and kidney (29b) concentration (PK
profiles; with values expressed in .mu.g/mL or .mu.g/gr) and
downregulation of TGF-.beta.2 mRNA (PD profile) in kidney following
single subcutaneous bolus administration of 50 mg/kg of
ASPH.sub.--0047 to Balb/c mice.
[0056] FIG. 30 depicts TGF-.beta.2 mRNA downregulation in
established subcutaneous tumors (FIG. 30A-D) or kidney (FIG. 30E-F)
in immunodeficient mouse following subcutaneous repeated
administration of ASPH.sub.--0047 or control oligonucleotide.
TGF-beta2 and GAPDH mRNA expression was detected by bDNA. Results
are expressed as TGF-beta2/GAPDH mRNA ratio, and each individual
tested sample is represented with line indicating median
values.
[0057] FIG. 31 shows the effect of systemic treatment of Balb/c
mice with ASPH.sub.--0047 (selective TGF-b2 antisense
oligonucleotide) on lung metastasis in orthotopic and in i.v. mouse
Renca renal carcinoma model. Level of lung metastasis was
determined by either number of metastasis or based on lung weight.
Results are shown as a box plot in which median values, upper and
lower quartiles, and 90th and 10th percentiles are presented.
[0058] FIG. 32 presents human Panc-1 pancreatic cancer cells were
treated with 3.3 .mu.M of the indicated oligonucleotides in the
absence of transfecting agent (gymnotic transfection or gymnotic
delivery). The expression of TGF-beta1 (black column), TGF-beta2
(white column) and TGF-beta3 (striped column) mRNA was determined
72 h after transfection.
[0059] FIG. 33 depicts the effect of systemic treatment of Balb/c
mice with ASPH.sub.--0047 on lung metastasis in orthotopic mouse
4T1 mammary carcinoma model. Data for each individual animal is
represented with median values indicated as bold black line.
DETAILED DESCRIPTION
[0060] The present invention is directed to oligonucleotides, in
particular antisense oligonucleotides, which comprise at least one
modified nucleotide and are suitable to interact with TGF-beta
mRNA. The oligonucleotides comprise or consist of 10 to 20, more
preferred 12 to 18 nucleotides of the TGF-beta2 nucleic acid
according to SEQ ID NO. 1 or of the TGF-beta1 nucleic acid
according to SEQ ID NO. 335, or of the nucleic acid sequence of
TGF-beta3 nucleic acid according to SEQ ID NO. 336. Most preferred
the oligonucleotide comprises or consists of 12, 13, 14, 15, 16,
17, or 18 nucleotides. The oligonucleotides are preferably selected
from the region of nucleic acid no. 1380 to 1510 (preferably no.
1380 to 1450 and/or no. 1480 to 1510), 1660 to 1680, or 2390 to
2410 of SEQ ID NO. 1. The oligonucleotide is a single or double
stranded RNA or DNA, including siRNA, microRNA, apatmer or
spiegelmer. Preferably, the oligonucleotide is an antisense
oligonucleotide.
[0061] A nucleotide forms the building block of an oligonucleotide,
and is for example composed of a nucleobase (nitrogenous base,
e.g., purine or pyrimidine), a five-carbon sugar (e.g., ribose,
2-deoxyribose, arabinose, xylose, lyxose, allose, altorse, glucose,
mannose, gulose, idose, galactose, talose or stabilized
modifications of those sugars), and one or more phosphate groups.
Examples of modified phosphate groups are phosphorothioate or
methylphosphonate. Each compound of the nucleotide is modifiable,
and is naturally or non-naturally occurring. The latter are for
example locked nucleic acid (LNA), a 2'-O,4'-C-ethylene-bridged
nucleic acid (ENA), polyalkylene oxide- (such as triethylene glycol
(TEG)), 2'-fluoro, 2'-O-methoxy and 2'-O-methyl modified
nucleotides as described for example by Freier & Altmann (Nucl.
Acid Res., 1997, 25, 4429-4443) and Uhlmann (Curr. Opinion in Drug
& Development (2000, 3 (2): 293-213), which are shown in FIG.
1.
[0062] A LNA is a modified RNA nucleotide, wherein the ribose
moiety is modified with an extra bridge connecting the 2' oxygen
and 4' carbon (2'-4'ribonucleoside). The bridge "locks" the ribose
in the 3'-endo (North) conformation, which is often found in the
A-form duplexes. LNA nucleosides and nucleotides, respectively,
comprise for example the forms of thio-LNA, oxy-LNA, or amino-LNA,
in alpha-D- or beta-L-configuration, and are mixable and
combineable, respectively, with DNA or RNA residues in the
oligonucleotide.
[0063] The oligonucleotides of the present invention, i.e.,
modified oligonucleotides, comprise at least one modified
nucleotide, preferably LNA and/or ENA, at the 5'- and/or 3'-end of
the oligonucleotide. In a preferred embodiment, the oligonucleotide
comprises 1, 2, 3, or 4 LNAs or ENAs at the 5'-end, and 1, 2, 3, or
4 LNAs or ENAs at the 3'-end. In another preferred embodiment, the
oligonucleotide comprises 1, 2, 3, or 4 LNAs or ENAs at the 5'-end
or 3'-end, and a polyalkylene oxide such as TEG at the 3'- or
5'-end. The modified oligonucleotides show a significantly
increased inhibition on TGF-beta expression and activity,
respectively, which results in an improved prevention and/or
treatment of a malignant or benign tumor, fibrosis (e.g.,
idiopathic pulmonary fibrosis, renal fibrosis, kidney fibrosis),
cirrhosis (e.g., liver cirrhosis), scleroderma or related
dermatologic diseases, an eye disease such as glaucoma or posterior
capsular opacification (PCO), a CNS disease, hair loss etc. The
oligonucleotides of the present invention target TGF-beta linked
diseases either by hybridization with TGF-beta mRNA, preferably
TGF-beta1, TGF-beta2, or TGF-beta3, alternatively, TGF-beta1,
TGF-beta2, and/or TGF-beta3 mRNAs, i.e., TGF-beta1 and TGF-beta2,
or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3, or
TGF-beta1, TGF-beta2 and TGF-beta3 mRNAs, or any other direct or
indirect effect on the TGF-beta system.
[0064] Preferably two or more oligonucleotides are combined,
wherein at least one oligonucleotide specifically inhibits
TGF-beta1 and at least one oligonucleotide specifically inhibits
TGF-beta2, or wherein at least one oligonucleotide specifically
inhibits TGF-beta1 and at least one oligonucleotide specifically
inhibits TGF-beta3, or wherein at least one oligonucleotide
specifically inhibits TGF-beta2 and at least one oligonucleotide
specifically inhibits TGF-beta3, or wherein at least one
oligonucleotide specifically inhibits TGF-beta1, at least one
oligonucleotide specifically inhibits TGF-beta2, and at least one
oligonucleotide specifically inhibits TGF-beta3.
[0065] In another embodiment, one oligonucleotide inhibits two
TGF-beta isoforms such as TGF-beta1 and TGF-beta2, TGF-beta2 and
TGF-beta3, or TGF-beta1 and TGF-beta3.
[0066] An oligonucleotide inhibiting the expression of all three
isoforms--TGF-beta1, TGF-beta2, and TGF-beta3--is defined as
pan-specific oligonucleotide.
[0067] In a further embodiment three or more oligonucleotides are
combined, wherein at least one oligonucleotide specifically
inhibits TGF-beta1, another oligonucleotide specifically inhibits
TGF-beta2, and a further oligonucleotide specifically inhibits
TGF-beta3, and optionally one or more additional oligonucleotides
inhibiting TGF-beta1, TGF-beta2 or TGF-beta3.
[0068] The oligonucleotides of the present invention have for
example an IC.sub.50 in the range of 0.1 to 20 .mu.M, preferably in
the range of 0.2 to 15 .mu.M, more preferably in the range of 0.4
to 10 .mu.M, and even more preferred in the range of 0.5 to 5
.mu.M.
[0069] The present invention further refers to a pharmaceutical
composition comprising an oligonucleotide according to the
invention as active ingredient. The pharmaceutical composition
comprises at least one oligonucleotide of the present invention and
optionally further an antisense compound, an antibody, a
chemotherapeutic compound, an anti-inflammatory compound, an
antiviral compound and/or an immuno-modulating compound.
Pharmaceutically acceptable binding agents and adjuvants or carrier
optionally comprise part of the pharmaceutical composition.
[0070] In one embodiment, the oligonucleotide and the
pharmaceutical composition, respectively, is formulated as dosage
unit in form of capsules, tablets and pills etc., respectively,
which contain for example the following compounds: microcrystalline
cellulose, gum or gelatin as binders; starch or lactose as
excipients; stearates as lubricants, various sweetening or
flavouring agents. For capsules the dosage unit may contain a
liquid carrier like fatty oils. Likewise coatings of sugar or
enteric agents may be part of the dosage unit.
[0071] The oligonucleotide and/or the pharmaceutical composition is
administrable via different routes. These routes of administration
include, but are not limited to, electroporation, epidermal,
impression into skin, intra-arterial, intra-articular,
intracranial, intradermal, intra-lesional, intra-muscular,
intranasal, intra-ocular, intrathecal, intracameral,
intraperitoneal, intraprostatic, intrapulmonary, intraspinal,
intratracheal, intratumoral, intravenous, intravesical, placement
within cavities of the body, nasal inhalation, oral, pulmonary
inhalation (e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer), subcutaneous, subdermal, topical
(including ophthalmic and to mucous membranes including vaginal and
rectal delivery), or transdermal administration.
[0072] For parenteral, subcutaneous, intradermal or topical
administration the oligonucleotide and/or the pharmaceutical
composition include for example a sterile diluent, buffers,
regulators of toxicity and antibacterials. In a preferred
embodiment, the oligonucleotide or pharmaceutical composition is
prepared with carriers that protect against degradation or
immediate elimination from the body, including implants or
microcapsules with controlled release properties. For intravenous
administration the preferred carriers are for example physiological
saline or phosphate buffered saline. An oligonucleotide and/or a
pharmaceutical composition comprising such oligonucleotide for oral
administration includes for example powder or granule,
microparticulate, nanoparticulate, suspension or solution in water
or non-aqueous media, capsule, gel capsule, sachet, tablet or
minitablet. An oligonucleotide and/or a pharmaceutical composition
comprising for parenteral, intrathecal, intracameral or
intraventricular administration includes for example sterile
aqueous solutions which optionally contain buffer, diluent and/or
other suitable additive such as penetration enhancer, carrier
compound and/or other pharmaceutically acceptable carrier or
excipient.
[0073] A pharmaceutically acceptable carrier is for example liquid
or solid, and is selected with the planned manner of administration
in mind so as to provide for the desired bulk, consistency, etc.,
when combined with a nucleic acid and the other components of a
given pharmaceutical composition. Typical pharmaceutically
acceptable carriers include, but are not limited to, a binding
agent (e.g. pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); filler (e.g. lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricant (e.g., magnesium stearate, talcum,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrate
(e.g., starch, sodium starch glycolate, etc.); or wetting agent
(e.g., sodium lauryl sulphate, etc.). Sustained release oral
delivery systems and/or enteric coatings for orally administered
dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552;
4,309,406; and 4,309,404. An adjuvant is included under these
phrases.
[0074] Beside being used in a method of human disease prevention
and/or treatment, the oligonucleotide and/or the pharmaceutical
composition according to the present invention is also used in a
method for prevention and/or treatment of other subjects including
veterinary animals, reptiles, birds, exotic animals and farm
animals, including mammals, rodents, and the like. Mammals include
for example horses, dogs, pigs, cats, or primates (for example, a
monkey, a chimpanzee, or a lemur). Rodents include for example
rats, rabbits, mice, squirrels, or guinea pigs.
[0075] The oligonucleotide or the pharmaceutical composition
according to the invention is used in a method for the prevention
and/or treatment of many different diseases, preferably benign or
malignant tumors, immunologic diseases, bronchial asthma, heart
disease, fibrosis (e.g., liver fibrosis, idiopathic pulmonary
fibrosis, liver cirrhosis, kidney cirrhosis, scleroderma),
diabetes, wound healing, disorders of the connective tissue (e.g.,
in heart, blood vessel, bone, joint, eye such as the Marfan or
Loeys-Dietz syndrome), psoriasis, eye diseases (e.g., glaucoma,
posterior capsular opacification (PCO) also known as secondary
cataract), CNS disease (e.g., Alzheimer's disease, Parkinson's
disease), coronary atherosclerosis (coronary intervention or
coronary artery bypass graft (CABG) surgery or hair loss. A tumor
is for example selected from the group of solid tumors, blood born
tumors, leukemias, tumor metastasis, hemangiomas, acoustic
neuromas, neurofibromas, trachomas, pyogenic granulomas,
astrocytoma such as anaplastic astrocytoma, acoustic neuroma,
blastoma, Ewing's tumor, craniopharyngloma, ependymoma,
medulloblastoma, glioma, glioblastoma, hemangloblastoma,
Hodgkins-lymphoma, medullablastoma, leukaemia, melanoma such as
primary and/or metastatic melanoma, mesothelioma, myeloma,
neuroblastoma, neurofibroma, non-Hodgkins lymphoma, pinealoma,
retinoblastoma, sarcoma, seminoma, trachomas, Wilm's tumor, bile
duct carcinoma, bladder carcinoma, brain tumor, breast cancer,
bronchogenic carcinoma, carcinoma of the kidney, cervical cancer,
choriocarcinoma, choroidcarcinoma, cystadenocarcinome, embryonal
carcinoma, epithelial carcinoma, esophageal cancer, cervical
carcinoma, colon carcinoma, colorectal carcinoma, endometrial
cancer, gallbladder cancer, gastric cancer, head cancer, liver
carcinoma, lung carcinoma, medullary carcinoma, neck cancer,
non-small-cell bronchogenic/lung carcinoma, ovarian cancer,
pancreas carcinoma, papillary carcinoma, papillary adenocarcinoma,
prostate cancer, small intestine carcinoma, prostate carcinoma,
rectal cancer, renal cell carcinoma (RCC, e.g., clear cell RCC,
papillary RCC, chromophobe RCC), oncocytoma kidney cancer,
transitional cell kidney cancer, retinoblastoma, skin cancer,
small-cell bronchogenic/lung carcinoma, squamous cell carcinoma,
sebaceous gland carcinoma, testicular carcinoma, and uterine
cancer. The oligonucleotide or the pharmaceutical composition of
the present invention is not only used in a method for the
prevention and/or treatment of a tumor, but likewise on a
metastasis.
[0076] The antisense oligonucleotides of the present invention are
characterized in that they show an unexpected low toxicity (see for
example Table 5) and thus, are well tolerated by different
organisms. They oligonucleotides show a reasonable distribution in
the organism, wherein highest concentrations are measured in the
kidney, liver, skin and spleen.
[0077] The present invention provides numerous oligonucleotides,
which are highly efficient in the reduction and inhibition,
respectively, of TGF-beta, in particular TGF-beta1, TGF-beta2
and/or TGF-beta3 expression due to the specific selection of the
sequence of the oligonucleotide and the modification of the
nucleotide. The following Table 1 shows numerous preferred modified
oligonucleotides according to the present invention (bold letters
indicate the modified nucleoside). Each oligonucleotides is defined
as ASPH and a number, which is defined by a specific sequence and
modification of the nucleosides:
TABLE-US-00001 SEQ ID NO. Sequence (5'->3') Modification ASPH 2
GACCAGATGCAGGA LNA 3 + 3 36 3 GCGACCGTGACCAGAT LNA 3 + 3 80 4
GCGCGACCGTGACC LNA 3 + 3 98 5 AGCGCGACCGTGA LNA 2 + 3 111 6
GACCGTGACCAGAT LNA 2 + 2 121 6 GACCGTGACCAGAT LNA 3 + TEG 153 7
CTGCCCGCGGAT LNA 2 + 2 15 8 TCTGCCCGCGGAT LNA 3 + 2 17 9
GGATCTGCCCGCGGA LNA 4 + 3 26 9 GGATCTGCCCGCGGA LNA 3 + 4 27 10
CTTGCTCAGGATCTGCC LNA 4 + 4 37 11 GCTCAGGATCTGCCCGCGGA 2' O-meth 4
+ 4 52 11 GCTCAGGATCTGCCCGCGGA 2' fluoro 4 + 4 53 12 GGATCGCCTCGAT
LNA 3 + 2 112 13 CCGCGGATCGCC LNA 2 + 2 119 14 ACCTCCTTGGCGTAGTA
LNA 3 + 3 01 14 ACCTCCTTGGCGTAGTA LNA 4 + 4 02 15 CCTCCTTGGCGTAGTA
LNA 3 + 3 03 15 CCTCCTTGGCGTAGTA LNA 4 + 4 04 16 CTCCTTGGCGTAGTA
LNA 3 + 3 05 16 CTCCTTGGCGTAGTA LNA 4 + 3 06 16 CTCCTTGGCGTAGTA LNA
3 + 4 07 17 TCCTTGGCGTAGTA LNA 3 + 3 08 18 CAGAAGTTGGCAT LNA 3 + 2
09 18 CAGAAGTTGGCAT LNA 2 + 3 10 19 AAGTGGGCGGGAT 11 19
AAGTGGGCGGGAT LNA 4 + 4 12 19 AAGTGGGCGGGAT 2' O-meth 4 + 4 13 19
AAGTGGGCGGGAT 2' fluoro 4 + 4 14 20 GCGGGATGGCAT LNA 2 + 2 16 21
GAAATCACCTCCG LNA 2 + 3 18 22 AAGTGGGCGGGAT LNA 2 + 3 19 23
TGTAGCGCTGGGT LNA 2 + 3 20 24 CGAAGGAGAGCCA LNA 3 + 2 21 25
TCGCGCTCGCAGGC LNA 3 + 3 22 26 AAGTGGGCGGGATG LNA 3 + 3 23 27
ATGTAGCGCTGGGT LNA 3 + 3 24 28 CGAAGGAGAGCCAT LNA 3 + 3 25 29
GAAAGTGGGCGGGAT LNA 4 + 3 28 30 CGAAGGAGAGCCATT LNA 4 + 3 29 31
CGATCCTCTTGCGCAT LNA 4 + 4 30 32 AAGTGGGCGGGATGGC LNA 4 + 4 31 33
GATGGAAATCACCTCCG LNA 4 + 4 32 34 AAACCTCCTTGGCGTAG LNA 4 + 4 33 35
TAGAAAGTGGGCGGGAT LNA 4 + 4 34 36 GGCGGGATGGCAT LNA 2 + 3 35 37
GGGTCTGTAGAAAGTG LNA 4 + 4 38 38 GAAGGAGAGCCATTC LNA 3 + 4 39 39
CCAGGTTCCTGTCTT LNA 3 + 4 40 40 TCTGATCACCACTGG LNA 3 + 4 41 41
TTTCTGATCACCACTGG LNA 4 + 4 42 42 GTCTGTAGGAGGGCA LNA 4 + 3 43 43
AGTCTGTAGGAGGGCA LNA 4 + 4 44 44 TCTGTAGGAGGGC LNA 2 + 3 45 45
CAGATGCCAGTTTTAAC LNA 4 + 4 46 46 CAAAGTATTTGGTCTCC LNA 4 + 4 47 47
CCTTAAGCCATCCATGA LNA 4 + 4 48 48 GTACTGGCCAGCTAA LNA 4 + 3 49 49
GCCTCGATCCTCTTGCGCAT 2' O-meth 4 + 4 50 49 GCCTCGATCCTCTTGCGCAT 2'
fluoro 4 + 4 51 50 AAACCTCCTTGGCGTAGTAC 2' O-meth 4 + 4 54 50
AAACCTCCTTGGCGTAGTAC 2' fluoro 4 + 4 55 51 GAAAGTGGGCGGGATGGCAT 2'
O-meth 4 + 4 56 51 GAAAGTGGGCGGGATGGCAT 2' fluoro 4 + 4 57 52
GAATTGCTCGCTTAGGG LNA 3 + 3 60 53 CGTCGCGGTTGCGTTCA LNA 3 + 3 61 54
CGTGGCCTACACCCTGG LNA 3 + 3 62 55 TTCTAAAGCAATAGGCC LNA 3 + 3 63 56
AGAATGGTTAGAGGTTC LNA 3 + 3 64 57 TCTGAACTAGTACCGCC LNA 3 + 3 65 58
CCCATTAATATGACCTC LNA 3 + 3 66 59 TTTAGTTAGAACCCTAA LNA 3 + 3 67 60
CCTCAGATATAGATAAC LNA 3 + 3 68 61 TACTATTATGGCATCCC LNA 3 + 3 69 62
TGCCCACTTGCATACTA LNA 3 + 3 70 63 AGCGTAATTGGTCATCA LNA 3 + 3 71 64
CGTTGGCAGAACATAGA LNA 3 + 3 72 65 GGGATACTGTCTAGACC LNA 3 + 3 73 66
ATTGGCAACTCGTTTGA LNA 3 + 3 74 67 CGTCAGGCTAATATTC LNA 3 + 3 75 68
GGATGACTCCCTAGAC LNA 3 + 3 76 69 GTCGCGGTTGCGTTCA LNA 3 + 3 77 70
CTCGGTACTCGGTCGG LNA 3 + 3 78 71 GGTTCGGTCCTGCCTT LNA 3 + 3 79 72
AATAGGCCGCATCCAA LNA 3 + 3 81 73 AACTAGTACCGCCTTT LNA 3 + 3 82 74
TCGGTCATATAATAAC LNA 3 + 3 83 75 AGACCGTCAGGCTAA LNA 3 + 3 84 76
GTCGCGGTTGCGTTC LNA 3 + 3 85 77 TTCCACTGCGGCGCT LNA 3 + 3 86 78
AAGGAGCGGTTCGGT LNA 3 + 3 87 79 CTCGGGTGCGGAGTG LNA 3 + 3 88 80
CTGACTTTGGCGAGT LNA 3 + 3 89 81 GATAGGAACGGTACG LNA 3 + 3 90 82
CACTTTGGATTCCCG LNA 3 + 3 91 83 GTCGCGGTTGCGTT LNA 3 + 3 92 84
TACACCCTGGCGGG LNA 3 + 3 93 85 CTCGGTACTCGGTC LNA 3 + 3 94 86
AGGAGCGGTTCGGT LNA 3 + 3 95 87 GTCTCGGGTGCGGA LNA 3 + 3 96 88
TACGGGACGGGCAG LNA 3 + 3 97 89 CGTCGCTCCTCTCG LNA 3 + 3 99 90
TAGCGCTGGGTTGG LNA 3 + 3 100 91 AAGCAATAGGCCGC LNA 3 + 3 101 92
TACGGGCATGCTCC LNA 3 + 3 102 93 AGGCGCGGGATAGG LNA 3 + 3 103 94
TTTGGATTCCCGCC LNA 3 + 3 104 95 ACCACTAGAGCACC LNA 3 + 3 105 96
GCGTTGGCAGAACA LNA 3 + 3 106 97 TTGCTCGCTTAGG LNA 2 + 3 107 98
GTCGCGGTTGCGT LNA 3 + 2 108 99 GGCGCTCGGTACT LNA 2 + 3 109 100
ATCTGAACTCGGC LNA 3 + 2 110 101 CGGTTGGTCTGTT LNA 2 + 3 113 102
TCCACCCTAGATC LNA 2 + 3 114 103 CTAGTACCGCCTT LNA 2 + 3 115 104
GGTCGGCAGTCAA LNA 3 + 2 116 105 CTTGCGACACCC LNA 2 + 2 117 106
GAGCGGTTCGGT LNA 2 + 2 118 107 ACACAGTAGTGCAT LNA 2 + 2 120 108
GGGTCTGTAGAAAG LNA 2 + 2 122 108 GGGTCTGTAGAAAG LNA 3 + TEG 154
109 GGTTGGAGATGTTA LNA 2 + 2 123 109 GGTTGGAGATGTTA LNA 3 + TEG 155
110 TGGGTTGGAGATGT LNA 2 + 2 124 110 TGGGTTGGAGATGT LNA 3 + TEG 156
111 GCTGGGTTGGAGAT LNA 2 + 2 125 111 GCTGGGTTGGAGAT LNA 3 + TEG 157
112 GCGCTGGGTTGGAG LNA 2 + 2 126 112 GCGCTGGGTTGGAG LNA 3 + TEG 158
113 AGCGCTGGGTTGGA LNA 2 + 2 127 113 AGCGCTGGGTTGGA LNA 3 + TEG 159
114 TAGCGCTGGGTTGG LNA 2 + 2 128 114 TAGCGCTGGGTTGG LNA 3 + TEG 160
115 GTAGCGCTGGGTTG LNA 2 + 2 129 115 GTAGCGCTGGGTTG LNA 3 + TEG 161
116 GATGTAGCGCTGGG LNA 2 + 2 130 116 GATGTAGCGCTGGG LNA 3 + TEG 162
117 CCATTCGCCTTCTG LNA 2 + 2 131 117 CCATTCGCCTTCTG LNA 3 + TEG 163
118 GAGAGCCATTCGCC LNA 2 + 2 132 118 GAGAGCCATTCGCC LNA 3 + TEG 164
119 AGCAGGGACAGTGT LNA 2 + 2 133 119 AGCAGGGACAGTGT LNA 3 + TEG 165
120 GCAGGAGATGTGGG LNA 2 + 2 134 120 GCAGGAGATGTGGG LNA 3 + TEG 166
121 CGGTTGGTCTGTTG LNA 2 + 2 135 121 CGGTTGGTCTGTTG LNA 3 + TEG 167
122 CCGGTTGGTCTGTT LNA 2 + 2 136 122 CCGGTTGGTCTGTT LNA 3 + TEG 168
123 GCCGGTTGGTCTGT LNA 2 + 2 137 123 GCCGGTTGGTCTGT LNA 3 + TEG 169
124 AGTTGGCATTGTAC LNA 2 + 2 138 124 AGTTGGCATTGTAC LNA 3 + TEG 170
125 GGTTAGAGGTTCTA LNA 2 + 2 139 125 GGTTAGAGGTTCTA LNA 3 + TEG 171
126 ATGGTTAGAGGTTC LNA 2 + 2 140 126 ATGGTTAGAGGTTC LNA 3 + TEG 172
127 AGAATGGTTAGAGG LNA 2 + 2 141 127 AGAATGGTTAGAGG LNA 3 + TEG 173
128 AGAGAATGGTTAGA LNA 2 + 2 142 128 AGAGAATGGTTAGA LNA 3 + TEG 174
129 CGTTGTCGTCGTCA LNA 2 + 2 143 129 CGTTGTCGTCGTCA LNA 3 + TEG 175
130 ACCAAGGCTCTCTT LNA 2 + 2 144 130 ACCAAGGCTCTCTT LNA 3 + TEG 176
131 GCTTCTTGTCTCTC LNA 2 + 2 145 131 GCTTCTTGTCTCTC LNA 3 + TEG 177
132 GGAACGGTACGTAC LNA 2 + 2 146 132 GGAACGGTACGTAC LNA 3 + TEG 178
133 TAGGAACGGTACGT LNA 2 + 2 147 133 TAGGAACGGTACGT LNA 3 + TEG 179
134 GGGATAGGAACGGT LNA 2 + 2 148 134 GGGATAGGAACGGT LNA 3 + TEG 180
135 CGCGGGATAGGAAC LNA 2 + 2 149 135 CGCGGGATAGGAAC LNA 3 + TEG 181
136 AGGCGCGGGATAGG LNA 2 + 2 150 136 AGGCGCGGGATAGG LNA 3 + TEG 182
137 GTCAAGCTGGATGG LNA 2 + 2 151 137 GTCAAGCTGGATGG LNA 3 + TEG 183
138 TCTGTAGGAGGGC ENA 2 + 3 184 139 GACCAGATGCAGGA ENA 3 + 3 185
140 CTCCTTGGCGTAGTA ENA 3 + 3 186 141 CCTCCTTGGCGTAGTA ENA 3 + 3
187 142 CAGATGCCAGTTTTAAC ENA 4 + 4 188 143 AGCGTAATTGGTCATCA ENA 3
+ 3 189 146 AGTATTTGGTCTCC LNA 3 + 3 190 147 AAGTATTTGGTCTC LNA 3 +
3 191 148 AAGTATTTGGTCTCC LNA 3 + 3 192 149 CAAAGTATTTGGTCTCC LNA 3
+ 3 193 150 AGCTCGTCCCTCCTCCC LNA 3 + 3 1000 151 GAGGGCTGGTCCGGAAT
LNA 3 + 3 1001 152 CGAGGGCTGGTCCGGAA LNA 3 + 3 1002 153
GAGGGCGGCATGGGGGA LNA 3 + 3 1003 154 GCGGGTGCTGTTGTACA LNA 3 + 3
1004 155 CGCGGGTGCTGTTGTAC LNA 3 + 3 1005 156 GTCGCGGGTGCTGTTGT LNA
3 + 3 1006 157 GGTCGCGGGTGCTGTTG LNA 3 + 3 1007 158
CCGGTCGCGGGTGCTGT LNA 3 + 3 1008 159 CCCGGTCGCGGGTGCTG LNA 3 + 3
1009 160 AGCACGCGGGTGACCTC LNA 3 + 3 1010 161 TTAGCACGCGGGTGACC LNA
3 + 3 1011 162 GGGCTCGTGGATCCACT LNA 3 + 3 1012 163
CCTTGGGCTCGTGGATC LNA 3 + 3 1013 164 TGGCATGGTAGCCCTTG LNA 3 + 3
1014 165 CGAGGGCTGGTCCGGA LNA 3 + 3 1015 166 GCGGGTGCTGTTGTAC LNA 3
+ 3 1016 167 GCACGCGGGTGACCTC LNA 3 + 3 1017 168 CCTTGGGCTCGTGGAT
LNA 3 + 3 1018 169 GGCATGGTAGCCCTTG LNA 3 + 3 1019 170
GGGTGCTGTTGTAC LNA 3 + 3 1020 171 TCGCGGGTGCTGTT LNA 3 + 3 1021 172
GTCGCGGGTGCTGT LNA 3 + 3 1022 173 CTCGTGGATCCACT LNA 3 + 3 1023 174
ATGGTAGCCCTTGG LNA 3 + 3 1024 175 TGGCATGGTAGCCC LNA 3 + 3 1025 176
GAAGTTGGCATGGT LNA 3 + 3 1026 177 TCGCGGGTGCTGT LNA 2 + 3 1027 178
CACCCGGTCGCGG LNA 2 + 3 1028 179 CCACCCGGTCGCG LNA 2 + 3 1029 180
CGCCAGGAATTGT LNA 3 + 2 1030 181 GGCTCGTGGATCC LNA 2 + 3 1031 182
TGGGCTCGTGGAT LNA 2 + 3 1032 183 GCATGGTAGCCCT LNA 2 + 3 1033 184
AGTTGGCATGGTA LNA 2 + 3 1034 185 TTGCAGGAGCGCA LNA 2 + 3 1035 186
ATTAGCACGCGGGTGAC LNA 3 + 3 1036 187 ACCATTAGCACGCGGGT LNA 3 + 3
1037 188 CACCATTAGCACGCGGG LNA 3 + 3 1038 189 CCACCATTAGCACGCGG LNA
3 + 3 1039 190 TCCACCATTAGCACGCG LNA 3 + 3 1040 191
TCCACCTTGGGCTTGCG LNA 3 + 3 1041 192 TTAGCACGCGGGTGAC LNA 3 + 3
1042 193 ACCATTAGCACGCGGG LNA 3 + 3 1043 194 CACCATTAGCACGCGG LNA 3
+ 3 1044 195 CACCATTAGCACGCG LNA 3 + 3 1045 196 GCGGCACGCAGCACG LNA
3 + 3 1046 197 TCGATGCGCTTCCG LNA 3 + 3 1047 198 TAGCACGCGGGTGA LNA
3 + 3 1048 199 ATTAGCACGCGGGT LNA 3 + 3 1049 200 CATTAGCACGCGGG LNA
3 + 3 1050 201 ACCATTAGCACGCG LNA 3 + 3 1051 202 CACCATTAGCACGC LNA
3 + 3 1052 203 CCACCATTAGCACG LNA 3 + 3 1053 204 TCCACCATTAGCAC LNA
3 + 3 1054 205 GACCTTGCTGTACT LNA 3 + 3 1055 206 GGACCTTGCTGTAC LNA
3 + 3 1056 207 AGGACCTTGCTGTA LNA 3 + 3 1057
208 CGGCACGCAGCACG LNA 3 + 3 1058 209 ACCTTGGGCTTGCG LNA 3 + 3 1059
210 TTAGCACGCGGGT LNA 3 + 2 1060 211 ACCATTAGCACGC LNA 3 + 2 1061
212 CGGCACGCAGCAC LNA 3 + 2 1062 213 CACCAGCTCCATGTCGA LNA 3 + 3
1063 214 TCGCGGGTGCTGTTGTA LNA 3 + 3 1064 215 GTGTCCAGGCTCCAAAT LNA
3 + 3 1065 215 GTGTCCAGGCTCCAAAT LNA 4 + 2 1066 216
GCTCGTCCCTCCTCCC LNA 3 + 3 1067 217 ACCAGCTCGTCCCTCC LNA 3 + 3 1068
218 GGAGGCCCCGCCCCTG LNA 3 + 3 1069 219 CATGGGGGAGGCGGCG LNA 3 + 3
1070 219 CATGGGGGAGGCGGCG 3LNA + 9N + 1LNA + 1071 1N + 2LNA 220
ACCAGCTCCATGTCGA LNA 3 + 3 1072 221 GGTCGCGGGTGCTGTT LNA 3 + 3 1073
222 GGACCTTGCTGTACTG LNA 3 + 3 1074 222 GGACCTTGCTGTACTG LNA 4 + 2
1075 223 TCCACCTTGGGCTTGC LNA 3 + 3 1076 224 AGCTCGTCCCTCCTC LNA 3
+ 3 1077 225 CCAGCTCGTCCCTCC LNA 3 + 3 1078 226 GAGGGCTGGTCCGGA LNA
3 + 3 1079 227 TCCCGAGGGCTGGTC LNA 3 + 3 1080 228 CGGCATGGGGGAGGC
LNA 2 + 4 1081 229 CAGCTCCATGTCGAT LNA 3 + 3 1082 230
ACCAGCTCCATGTCG LNA 3 + 3 1083 231 TCGCGGGTGCTGTTG LNA 3 + 3 1084
232 GTCGCGGGTGCTGTT LNA 3 + 3 1085 233 GGTCGCGGGTGCTGT LNA 3 + 3
1086 234 AGCACGCGGGTGACC LNA 3 + 3 1087 235 TAGCACGCGGGTGAC LNA 3 +
3 1088 236 CATTAGCACGCGGGT LNA 3 + 3 1089 237 TCCACCATTAGCACG LNA 3
+ 3 1090 238 CCAGGAATTGTTGCT LNA 4 + 2 1091 239 TTGGGCTCGTGGATC LNA
3 + 3 1092 240 CTTGGGCTCGTGGAT LNA 3 + 3 1093 241 TTGGCATGGTAGCCC
LNA 3 + 3 1094 242 GAAGTTGGCATGGTA LNA 3 + 3 1095 243
AGAAGTTGGCATGGT LNA 3 + 3 1096 244 TGTCCAGGCTCCAAA LNA 4 + 2 1097
245 AGGACCTTGCTGTAC LNA 3 + 3 1098 246 CACCTTGGGCTTGCG LNA 4 + 2
1099 246 CACCTTGGGCTTGCG 1LNA + 1N + 2LNA + 1100 8N + 1LNA + 1N +
1LNA 247 AGCTCGTCCCTCCT LNA 3 + 3 1101 248 CAGCTCGTCCCTCC LNA 3 + 3
1102 249 ACCAGCTCGTCCCT LNA 3 + 3 1103 250 CCCGAGGGCTGGTC LNA 3 + 3
1104 251 GCGGCATGGGGGAG LNA 2 + 4 1105 252 GTCTTGCAGGTGGA LNA 3 + 3
1106 253 TCGATGCGCTTCCG LNA 2 + 4 1107 253 TCGATGCGCTTCCG LNA 2 + 3
1108 253 TCGATGCGCTTCCG 2LNA + 8N + 2LNA + 1109 1N + 1LNA 253
TCGATGCGCTTCCG 2LNA + 9N + 1LNA + 1110 1N + 1LNA 253 TCGATGCGCTTCCG
2LNA + 8N + 1LNA + 1111 2N + 1LNA 254 GGACAGGATCTGGC LNA 4 + 2 1112
255 ACCTCCCCCTGGCT LNA 3 + 3 1113 256 ACCATTAGCACGCG LNA 4 + 2 1114
256 ACCATTAGCACGCG 3LNA + 8N + 1LNA + 1115 1N + 1LNA 257
CAGCAGTTCTTCTC LNA 2 + 4 1116 258 TACAGCTGCCGCAC LNA 3 + 3 1117 259
AGTTGGCATGGTAG LNA 3 + 3 1118 259 AGTTGGCATGGTAG LNA 4 + 2 1119 260
AAGTTGGCATGGTA LNA 3 + 3 1120 261 GAAGTTGGCATGGT LNA 4 + 2 1121 262
TCCAGGCTCCAAAT LNA 3 + 3 1122 263 ACCTTGCTGTACTG LNA 3 + 3 1123 263
ACCTTGGGCTTGCG LNA 4 + 2 1124 263 ACCTTGGGCTTGCG LNA 3 + 2 1125 263
ACCTTGGGCTTGCG 3LNA + 8N + 1LNA + 1126 1N + 1LNA 263 ACCTTGGGCTTGCG
2LNA + 9N + 1LNA + 1127 1N + 1LNA 263 ACCTTGGGCTTGCG 2LNA + 8N +
2LNA + 1128 1N + 1LNA 264 TTGCAGGAGCGCAC LNA 3 + 3 1129 265
GCAGAAGTTGGCAT LNA 4 + 2 1130 266 CGGGTGCTGTTGTA LNA 3 + 3 1131 266
CGGGTGCTGTTGTA LNA 2 + 4 1132 267 CCCAGCGGCAACGGAAA LNA 3 + 3 1133
268 CAAGAGGTCCCCGCGCC LNA 3 + 3 1134 269 GCGTCCCCGGCGGCAAA LNA 3 +
3 1135 270 GGTCGGCGACTCCCGAG LNA 3 + 3 1136 271 TCGGAGAGAGATCCGTC
LNA 3 + 3 1137 272 ATCCCACGGAAATAACC LNA 3 + 3 1138 273
CTCAGTATCCCACGGAA LNA 3 + 3 1139 274 ACTGCCGAGAGCGCGAA LNA 3 + 3
1140 275 CTGATGTGTTGAAGAAC LNA 3 + 3 1141 276 TGAGGTATCGCCAGGAA LNA
3 + 3 1142 277 ACTGCCGCACAACTCCG LNA 3 + 3 1143 278
CGGCCCACGTAGTACAC LNA 3 + 3 1144 279 CCCAGCGGCAACGGAA LNA 3 + 3
1145 280 TCGCGCCAAGAGGTCC LNA 3 + 3 1146 281 GGTCGGCGACTCCCGA LNA 3
+ 3 1147 282 GTCGGAGAGAGATCCG LNA 3 + 3 1148 283 TCAGTATCCCACGGAA
LNA 3 + 3 1149 284 CGAGAGCGCGAACAGG LNA 3 + 3 1150 285
ACTGCCGAGAGCGCGA LNA 3 + 3 1151 286 GGCGTCAGCACCAGTA LNA 3 + 3 1152
287 GGTTTCCACCATTAGC LNA 3 + 3 1153 288 GAGGTATCGCCAGGAA LNA 3 + 3
1154 289 AACCACTGCCGCACAA LNA 3 + 3 1155 290 CGGCCCACGTAGTACA LNA 3
+ 3 1156 291 CGGCGGCTCGTCTCA LNA 3 + 3 1157 292 CCCAGCGGCAACGGA LNA
3 + 3 1158 293 TCGCGCCAAGAGGTC LNA 3 + 3 1159 294 CGTCGCGCCAAGAGG
LNA 3 + 3 1160 295 GGAGCAAGCGTCCCC LNA 3 + 3 1161 296
GTGCGCCCGAGGTCT LNA 3 + 3 1162 297 GTCTAGGATGCGCGG LNA 3 + 3 1163
298 CAGTATCCCACGGAA LNA 3 + 3 1164 299 CCGAGAGCGCGAACA LNA 3 + 3
1165 300 GGCGTCAGCACCAGT LNA 3 + 3 1166 301 GTTGCTGAGGTATCG LNA 3 +
3 1167 302 ACCACTGCCGCACAA LNA 3 + 3 1168 303 CGGCCCACGTAGTAC LNA 3
+ 3 1169 304 CTCGGCGACTCCTT LNA 3 + 3 1170 305 AGCGGCAACGGAAA LNA 3
+ 3 1171 306 TCGCGCCAAGAGGT LNA 3 + 3 1172 307 TCCCCGGCGGCAAA LNA 3
+ 3 1173 308 TGCGCCCGAGGTCT LNA 3 + 3 1174 309 GTCTAGGATGCGCG LNA 3
+ 3 1175 310 GGTCGGAGAGAGAT LNA 3 + 3 1176 311 CACGGAAATAACCT LNA 3
+ 3 1177 312 AGAGCGCGAACAGG LNA 3 + 3 1178
313 ATAGTCCCGCGGCC LNA 3 + 3 1179 314 TAGTAGTCGGCCTC LNA 3 + 3 1180
315 ATAGATTTCGTTGT LNA 3 + 3 1181 316 GAGGTATCGCCAGG LNA 3 + 3 1182
317 GCCGCACAACTCCG LNA 3 + 3 1183 318 TCGCGCCAAGAGG LNA 2 + 3 1184
319 AAGCGTCCCCGGC LNA 3 + 2 1185 320 GACGCCGTGTAGG LNA 3 + 2 1186
321 GTCGGCGACTCCC LNA 2 + 3 1187 322 TGCGCCCGAGGTC LNA 3 + 2 1188
323 GTCGGAGAGAGAT LNA 3 + 2 1189 324 TCCCACGGAAATA LNA 3 + 2 1190
325 TGCCGAGAGCGCG LNA 2 + 3 1191 326 TAGTCCCGCGGCC LNA 3 + 2 1192
327 TAGTAGTCGGCCT LNA 3 + 2 1193 328 CATAGATTTCGTT LNA 2 + 3 1194
329 TTTAACTTGAGCC LNA 3 + 2 1195 330 GAGGTATCGCCAG LNA 3 + 2 1196
331 ACTCCGGTGACAT LNA 2 + 3 1197 332 GCCCACGTAGTAC LNA 2 + 3 1198
333 TCGGCGACTCCC LNA 2 + 2 1199 334 GTCGGCGACTCC LNA 2 + 2 1200 337
CAGGAAGCGCTGGCAAC LNA 3 + 3 2000 338 GGTGCATGAACTCACTG LNA 3 + 3
2001 339 GTCCCCTAATGGCTTCC LNA 3 + 3 2002 340 ATCTGTCCCCTAATGGC LNA
3 + 3 2003 341 CCGGGTGCTGTTGTAAA LNA 3 + 3 2004 342
CCTGGATCATGTCGAAT LNA 3 + 3 2005 343 CCCTGGATCATGTCGAA LNA 3 + 3
2006 344 GTAGCACCTGCTTCCAG LNA 3 + 3 2007 345 GGGCTTTCTAAATGAC LNA
3 + 3 2008 346 TGACTCCCAGCAGGCC LNA 3 + 3 2009 347 GTGCATGAACTCACTG
LNA 3 + 3 2010 348 GGTGCATGAACTCACT LNA 3 + 3 2011 349
ATCTGTCCCCTAATGG LNA 3 + 3 2012 350 CGGGTGCTGTTGTAAA LNA 3 + 3 2013
351 CCGGGTGCTGTTGTAA LNA 3 + 3 2014 352 CCTGGATCATGTCGAA LNA 3 + 3
2015 353 CCCTGGATCATGTCGA LNA 3 + 3 2016 354 TTTGAATTTGATTTCC LNA 3
+ 3 2017 355 GGGCCTGAGCAGAAGT LNA 3 + 3 2018 356 GGGGGCTTTCTAAAT
LNA 3 + 3 2019 357 TTTGTTTACACTTCC LNA 3 + 3 2020 358
CCAGCTAAAGGTGGG LNA 3 + 3 2021 359 ATGGCTGGGTCCCAA LNA 3 + 3 2022
360 GAGTTTTTCCTTAGG LNA 3 + 3 2023 361 AGGGGTGGCAAGGCA LNA 3 + 3
2024 362 TGACTCCCAGCAGGC LNA 3 + 3 2025 363 GAAGCGCTGGCAACC LNA 3 +
3 2026 364 GTGCATGAACTCACT LNA 3 + 3 2027 365 GTGGTGCAAGTGGAC LNA 3
+ 3 2028 366 CTAATGGCTTCCACC LNA 3 + 3 2029 367 CCCCTAATGGCTTCC LNA
3 + 3 2030 368 ATCTGTCCCCTAATG LNA 3 + 3 2031 369 GATCTGTCCCCTAAT
LNA 3 + 3 2032 370 AGATCTGTCCCCTAA LNA 3 + 3 2033 371
GGTGCTGTTGTAAAG LNA 3 + 3 2034 372 CCGGGTGCTGTTGTA LNA 3 + 3 2035
373 GATCATGTCGAATTT LNA 3 + 3 2036 374 CCTGGATCATGTCGA LNA 3 + 3
2037 375 CCCTGGATCATGTCG LNA 3 + 3 2038 376 GATTTCCATCACCTC LNA 3 +
3 2039 377 TTGAATTTGATTTCC LNA 3 + 3 2040 378 AGCAGTTCTCCTCCA LNA 3
+ 3 2041 379 GCCTGAGCAGAAGTT LNA 3 + 3 2042 380 GGGCAAGGGCCTGAG LNA
3 + 3 2043 381 CCCACACTTTCTTTA LNA 3 + 3 2044 382 TAGCACCTGCTTCCA
LNA 3 + 3 2045 383 CGGGGGCTTTCTAA LNA 3 + 3 2046 384 CCATTCATGCTTTC
LNA 3 + 3 2047 385 AAGCGCTGGCAACC LNA 3 + 3 2048 386 ACCAGAGCCCTTTG
LNA 3 + 3 2049 387 CCCCTAATGGCTTC LNA 3 + 3 2050 388 GTCCCCTAATGGCT
LNA 3 + 3 2051 389 ATCTGCCCCTAAT LNA 3 + 3 2052 390 AGATCTGTCCCCTA
LNA 3 + 3 2053 391 CGGGTGCTGTTGTA LNA 3 + 3 2054 392 ATCATGTCGAATTT
LNA 3 + 3 2055 393 CCCTGGATCATGTC LNA 3 + 3 2056 394 CCTTTGAATTTGAT
LNA 3 + 3 2057 395 TTGCGGAAGCAGTA LNA 3 + 3 2058 396 GCCTGAGCAGAAGT
LNA 3 + 3 2059 397 GGGGGCTTTCTAA LNA 2 + 3 2060 398 AGCGCTGGCAACC
LNA 2 + 3 2061 399 CCCCTAATGGCTT LNA 2 + 3 2062 399 CCCCTAATGGCTT
LNA 3 + 2 2063 400 TCCCCTAATGGCT LNA 3 + 2 2064 401 TCATGTCGAATTT
LNA 2 + 3 2065 402 ATCATGTCGAATT LNA 3 + 2 2066
[0078] Table 1 shows the nucleic acid sequences of selected
oligonucleotides of the present invention as well as the
modifications of the nucleotides, wherein LNA 4+4 means
4.times.LNAs at the 5'- and 3'-end of the oligonucleotide are
modified, wherein LNA 4+3 means 4.times.LNAs at the 5'-end and
3.times.LNAs at the 3'-end of the oligonucleotide are modified,
wherein LNA 3+4 means 3.times.LNAs at the 5'-end and 4.times.LNAs
at the 3'-end of the oligonucleotide are modified, wherein LNA 3+3
means 3.times.LNAs at the 5'- and 3'-end of the oligonucleotide are
modified, wherein LNA 3+2 means 3.times.LNAs at the 5'-end and
2.times.LNAs at the 3'-end of the oligonucleotide are modified,
wherein LNA 2+3 means 2.times.LNAs at the 5'-end and 3.times.LNAs
at the 3'-end of the oligonucleotide are modified, wherein LNA 2+2
means 2.times.LNAs at the 5'- and 3'-end of the oligonucleotide are
modified. Alternatively, some oligonucleotides comprise ENA 4+4,
i.e., 4.times.ENA at the 5'- and 3'-end of the oligonucleotide are
modified, or ENA 3+3, i.e., 3.times.ENA at the 5'- and 3'-end of
the oligonucleotide are modified. Further oligonucleotides comprise
2' 0-meth 4+4, wherein the oligonucleotide comprises 4.times.2'
O-methyl modified nucleotides at the 5'- and 3'-end of the
oligonucleotide, or comprises 2' fluoro 4+4, wherein the
oligonucleotide comprises 4.times.2' fluoro modified nucleotides at
the 5'- and 3'-end. Oligonucleotides comprising LNA 3+TEG comprise
3.times.LNAs at the 5'-end and one triethylene glycol (TEG) at the
3'-end of the oligonucleotide. Some oligonucleotides comprise LNAs
which are not arranged in a row but are separated by an unlocked
nucleoside having for example the sequences 3LNA+9N+1LNA+1N+2LNA,
1LNA+1N+2LNA+8N+1LNA+1N+1LNA, 2LNA+8N+2LNA+1N+1LNA,
2LNA+9N+1LNA+1N+1LNA, 2LNA+8N+1LNA+2N+1LNA, 3LNA+8N+1LNA+1N+1LNA,
3LNA+8N+1LNA+1N+1LNA, 2LNA+9N+1LNA+1N+1LNA, or
2LNA+8N+2LNA+1N+1LNA, wherein "N" is a nucleoside without locked
modification. "ASPH" in combination with a number refers to the
different oligonucleotides and their different modifications as
described in Table 1. These modified oligonucleotides were tested
e.g. in experiments shown in the following examples. The antisense
oligonucleotides of the present invention can be described
differently, e.g., ASPH47, ASPH0047, ASPH.sub.--47 or
ASPH.sub.--0047 referring to the same oligonucleotide.
[0079] For the purpose of clarity and a concise description,
features are described herein as part of the same or separate
embodiments, however, it will be appreciated that the scope of the
invention may include embodiments having combinations of all or
some of the features described.
[0080] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that the scope of the present invention refers to
various other embodiments, modifications, and equivalents thereof
which, after reading the description herein, may suggest themselves
to those skilled in the art without departing from the spirit of
the invention.
EXAMPLES
[0081] In the following examples, the effect of the
oligonucleotides listed in Table 1 has been tested in view of the
reduction and inhibition, respectively, of TGF-beta1 and/or
TGF-beta2 expression. SEQ ID NO. 144 (T-LNA: CGGCATGTCTATTTTGTA,
wherein 3.times.nucleotides at the 5'- and 3'-end are LNAs) and SEQ
ID NO. 145 (scr-LNA: CGTTTAGGCTATGTACTT, wherein
3.times.nucleotides at the 5'- and 3'-end are LNAs) are used as
control oligonucleotides, wherein SEQ ID NO. 145 (negative control)
is the scrambled form of SEQ ID NO. 144 (positive control). The
cells were either transfected in the presence of a transfecting
agent (e.g., Lipofectamine), or in the absence of any transfecting
agent (which is defined as gymnotic transfection or unassisted
transfection or gymnotic delivery). In case of gymnotic delivery
the entry of the oligonucleotide into the cell solely depends on
the interaction of the oligonucleotide with the cell (no
compound/agent supports the entry). Therefore, gymnotic
transfection or gymnotic delivery is considered to reflect better
conditions of the in vivo settings.
Example 1
[0082] Human A172 glioma cells were transfected with 10 nM of
ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08,
ASPH09, ASPH10, ASPH11, ASPH12, ASPH13, ASPH14, ASPH15, ASPH16,
ASPH17, ASPH18, ASPH19, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25,
ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH34,
ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42,
ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50,
ASPH51, ASPH52, ASPH53, and ASPH54 (see FIG. 3a); ASPH36, ASPH60,
ASPH61, ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68,
ASPH69, ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76,
ASPH77, ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84,
ASPH85, ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92,
ASPH93, ASPH94, ASPH95, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100,
ASPH101, ASPH102, ASPH103, ASPH104, ASPH105, ASPH106, ASPH107,
ASPH108, ASPH109, ASPH110, ASPH111, ASPH112, ASPH113, ASPH114,
ASPH115, ASPH116, ASPH117, ASPH118, and ASPH119 (see FIG. 3b), or
ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122, ASPH123,
ASPH124, ASPH125, ASPH126, ASPH127, ASPH128, ASPH129, ASPH130,
ASPH131, ASPH132, ASPH133, ASPH134, ASPH135, ASPH136, ASPH137,
ASPH138, ASPH139, ASPH140, ASPH141, ASPH142, ASPH143, ASPH145,
ASPH146, ASPH147, ASPH148, ASPH149, ASPH150, ASPH151, ASPH152,
ASPH153, ASPH154, ASPH155, ASPH157, ASPH158, ASPH160, ASPH161,
ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168,
ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175,
ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and
ASPH183 (see FIG. 3c), and the controls of SEQ ID NO. 144 and 145,
respectively, in the presence of a transfecting agent. The
expression of TGF-beta1 and TGF-beta2 mRNA was determined 24 h
after transfection. Significant reduction of the expression of
TGF-beta1 and TGF-beta2 mRNA is demonstrated in FIGS. 3a) to 3c).
The dual TGF-beta1 and TGF-beta2 reactive oligonucleotides ASPH01,
ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08 and ASPH09
show a significant reduction of the expression of both TGF-beta1
and TGF-beta2 mRNA, while the selective TGF-beta2 oligonucleotides
significantly inhibit TGF-beta2 mRNA expression.
Example 2
[0083] Human Panc-1 pancreatic cancer cells were transfected with
10 nM of ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07,
ASPH08, ASPH12, ASPH14, ASPH17, ASPH18, ASPH20, ASPH21, ASPH22,
ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32,
ASPH33, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41,
ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49,
ASPH50, ASPH51, and ASPH52 (see FIG. 4a); ASPH36, ASPH60, ASPH61,
ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69,
ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77,
ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85,
ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93,
ASPH94, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102,
ASPH103, ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109,
ASPH110, ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116,
ASPH117, ASPH118, and ASPH119 (see FIG. 4b), or ASPH36, ASPH71,
ASPH73, ASPH120, ASPH121, ASPH122, ASPH127, ASPH128, ASPH129,
ASPH130, ASPH131, ASPH132, ASPH133, ASPH135, ASPH136, ASPH137,
ASPH139, ASPH141, ASPH142, ASPH143, ASPH145, ASPH146, ASPH147,
ASPH149, ASPH150, ASPH151, ASPH152, ASPH153, ASPH154, ASPH155,
ASPH157, ASPH160, ASPH161, ASPH162, ASPH163, ASPH164, ASPH165,
ASPH166, ASPH167, ASPH168, ASPH169, ASPH170, ASPH171, ASPH172,
ASPH173, ASPH174, ASPH175, ASPH176, ASPH177, ASPH178, ASPH179,
ASPH180, ASPH181, ASPH182, and ASPH183 (see FIG. 4c) and the
controls of SEQ ID NO. 144 and 145, respectively, in the presence
of a transfecting agent. The expression of TGF-beta1 and TGF-beta2
mRNA was determined 24 h after transfection. Significant reduction
of the expression of TGF-beta1 and TGF-beta2 mRNA is demonstrated
in FIGS. 4a) to 4c). The dual TGF-beta1 and TGF-beta2 reactive
oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06,
ASPH07, and ASPH08, respectively, show again a significant
reduction of the expression of both TGF-beta1 and TGF-beta2 mRNA,
while the selective TGF-beta2 oligonucleotides significantly
inhibit TGF-beta2 mRNA expression.
Example 3
[0084] In further experiments the inhibitory effect of each of
ASPH01, ASPH03, ASPH05, ASPH17, ASPH18, ASPH22, ASPH26, ASPH27,
ASPH33, ASPH36, ASPH37, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47,
ASPH48, ASPH49, ASPH64, ASPH65, ASPH66, ASPH69, ASPH71, ASPH80,
ASPH82, ASPH88, ASPH89, ASPH90, ASPH98, ASPH99, ASPH102, ASPH105,
ASPH115, ASPH121, ASPH140, ASPH153, ASPH165, ASPH171, ASPH178,
ASPH181, ASPH184, ASPH185, ASPH186, ASPH187, ASPH188, ASPH189, and
of the controls of SEQ ID NO.144 and SEQ ID NO. 145 was tested in
human A172 glioma cells. A172 cells were transfected with these
modified oligonucleotides in doses of 20 nM, 4 nM, 0.8 nM, 0.16 nM,
and 0.04 nM, respectively, in the presence of a transfecting agent.
The remaining TGF-beta2 mRNA was measured 24 h after transfection.
TGF-beta2 values were normalized to GAPDH and oligonucleotide
concentrations resulting in 50% reduction of TGF-beta2 mRNA
(=IC.sub.50 values) were calculated. All IC.sub.50 values were
referenced to the IC.sub.50 value of ASPH.sub.--036 (ASPH36) that
was 0.33 nM and the results are shown as fold-difference of the
IC.sub.50 value of ASPH.sub.--036 Table 2:
TABLE-US-00002 Fold IC.sub.50 referenced Oligonucleotide to
ASPH_036 ASPH_080 0.591 ASPH_069 0.673 ASPH_065 0.773 ASPH_105
0.882 ASPH_036 1.000 ASPH_046 1.142 ASPH_098 1.182 ASPH_071 1.237
ASPH_026 1.242 ASPH_047 1.303 ASPH_088 1.455 ASPH_185 1.456
ASPH_115 1.545 ASPH_153 1.665 ASPH_181 1.918 ASPH_027 2.000
ASPH_089 2.091 ASPH_102 2.091 ASPH_041 2.182 ASPH_018 2.212
ASPH_049 2.455 ASPH_022 2.485 ASPH_188 2.639 ASPH_189 2.660
ASPH_042 2.848 ASPH_178 3.147 ASPH_048 3.182 ASPH_066 3.182
ASPH_033 3.182 ASPH_045 3.636 ASPH_121 3.644 ASPH_171 3.871
ASPH_005 3.954 ASPH_003 4.111 ASPH_082 4.818 ASPH_037 5.303
ASPH_099 5.545 ASPH_090 6.727 ASPH_165 7.175 ASPH_186 7.655
ASPH_017 8.455 ASPH_001 9.242 ASPH_187 9.990 ASPH_064 10.091
ASPH_140 11.482 ASPH_184 12.224 SEQ ID NO 144 17.212 SEQ ID NO 145
n.a
[0085] All the modified oligonucleotides show an IC.sub.50 in a low
nanomolar to picomolar range, which is markedly lower than
IC.sub.50 of the positive control oligonucleotide of SEQ ID NO.
144; the IC.sub.50 of the negative control of SEQ ID NO. 145 was
not calculable.
Example 4
[0086] Human Panc-1 pancreatic cancer cells were treated with 3.3
.mu.M of ASPH17, ASPH18, ASPH22, ASPH25, ASPH33, ASPH35, ASPH36,
ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH65,
ASPH66, ASPH67, ASPH69, ASPH71, ASPH79, ASPH80, ASPH82, ASPH88,
ASPH89, ASPH90, ASPH91, ASPH98, ASPH99, ASPH102, ASPH105, ASPH111,
ASPH115, ASPH119, ASPH121, ASPH139, ASPH140, ASPH146, ASPH151,
ASPH153, ASPH165, ASPH171, ASPH172, ASPH176, ASPH178, ASPH180, and
ASPH183, respectively, or the controls of SEQ ID NO. 144 and 145 in
the absence of a transfecting agent (gymnotic transfection or
gymnotic delivery). The inhibitory effect of the modified
oligonucleotides on expression of TGF-beta1 and TGF-beta2 mRNA,
respectively, was determined 72 h after treatment start. Under
gymnotic delivery experimental conditions, the oligonucleotides
enter the cells and strongly inhibit the expression of TGF-beta2
mRNA. The results of the experiments are shown in FIG. 5.
Example 5
[0087] In further experiments human Panc-1 pancreatic cancer cells
were transfected with 10 .mu.M of modified oligonucleotides ASPH01,
ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH35, ASPH36,
ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, and ASPH48, respectively,
or the controls of SEQ ID NO. 144 and 145 in the absence of a
transfecting agent (gymnotic transfection or gymnotic delivery).
The oligonucleotides were added to the cells for 2 days, after
which medium was changed, and further incubation for 2 days was
carried out in oligonucleotide-containing medium. Expression of
TGF-beta1 mRNA (FIG. 6a) and TGF-beta2 mRNA (FIG. 6b) was then
measured and normalized to HPRT1
(Hypoxanthin-Phosphoribosyl-Transferase1). Cell supernatants were
analysed for TGF-beta1 (FIG. 7a) and TGF-beta2 (FIG. 7b) protein by
ELISA. Under gymnotic delivery experimental conditions, dual
TGF-beta1 and TGF-beta2 reactive oligonucleotide ASPH01, ASPH03,
ASPH05, and pan-specific ASPH09 significantly inhibit the
expression of TGF-beta1 and TGF-beta2 mRNA, and protein. All the
other oligonucleotides significantly inhibit the expression of
TGF-beta2 mRNA and protein.
Example 6
[0088] In another experiment dose dependency of the inhibitory
effect of modified oligonucleotides of the present invention was
tested. Human Panc-1 pancreatic cancer cells were treated with 15
.mu.M, 10 .mu.M, 7.5 .mu.M, 5 .mu.M, 2.5 .mu.M, 1.25 .mu.M, or
0.625 .mu.M ASPH05 or ASPH36, or the controls of SEQ ID NO. 144 and
145, respectively, without using a transfection reagent. The
oligonucleotides were added to the cells for 2 days. Thereafter
media were changed and cells were incubated for 2 further days in
oligonucleotide-containing medium, after which (total treatment
time: 4 days) the expression of TGF-beta1 (FIG. 8a) and TGF-beta2
(FIG. 8b) mRNA was measured. The dual TGF-beta1 and TGF-beta2
reactive oligonucleotide ASPH05 shows a marked dose dependent
inhibition of both TGF-beta1 and TGF-beta2 mRNA expression, and
ASPH36 inhibits specifically the expression of TGF-beta2 mRNA in a
dose-dependent manner.
Example 7
[0089] Mouse SMA-560 glioma cells were transfected with 10 nM
ASPH01, ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH26,
ASPH36, ASPH37, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, or ASPH48,
or the controls of SEQ ID NO. 144 and 145, respectively, in the
presence of a transfecting agent. 24 h after transfection, the
inhibition of the expression of TGF-beta1 (white columns) and
TGF-beta2 (black columns) mRNA was determined. The pan-specific
ASPH09 inhibits the expression of the mouse TGF-beta1 mRNA, and the
other oligonucleotides tested strongly inhibit the expression of
the mouse TGF-beta2 mRNA. The results are presented in FIG. 9.
Example 8
[0090] Female athymic nude mice (Hsd:Athymic Nude-Foxn1.sup.nu)
were treated for 5 consecutive days with 14 mg/kg or 50 mg/kg of
oligonucleotide ASPH01, ASPH03, ASPH05, ASPH17, ASPH22, ASPH37,
ASPH41, ASPH45, ASPH46, ASPH47, or ASPH48, and control of SEQ ID
NO. 145, respectively, or saline by subcutaneous injection. The day
after the last treatment, the mice were sacrificed. Mouse TGF-beta2
mRNA was quantified in kidney tissue lysates. In FIG. 10,
data--representing TGF-beta2 to GAPDH mRNA ratio--are shown as a
box plot in which median values and min. and max. values are
presented (data expressed as n=4, except ASPH46 group n=3). All the
tested oligonucleotides inhibited the expression of TGF-beta2 mRNA
in the kidney of these mice.
Example 9
[0091] In another experiment human Panc-1 pancreatic cancer cells
were transfected with 10 .mu.M of modified oligonucleotide ASPH09
or the control of SEQ ID NO. 145 in the absence of any transfecting
agent (gymnotic transfection or gymnotic delivery). The
oligonucleotides were added to the cells for 2 days, after medium
was changed, and further incubation for 2 days was carried out in
oligonucleotide-containing medium. Expression of TGF-beta3 mRNA
(see FIG. 11) was then measured and normalized to HPRT1
(Hypoxanthin-Phosphoribosyl-Transferase1). Under gymnotic delivery
experimental conditions, the pan-specific oligonucleotide ASPH09
significantly inhibits the expression of TGF-beta3 mRNA.
Example 10
[0092] Human Panc-1 pancreatic cancer cells were treated with 10
.mu.M, 3.3 .mu.M, 1.1 .mu.M, 0.37 .mu.M, and 0.12 .mu.M of ASPH03,
ASPH36, ASPH45, ASPH47, ASPH65, ASPH69, AASPH71, ASPH80, ASPH115,
ASPH 121, ASPH153, ASPH185, and ASPH189, respectively, in the
absence of a transfecting agent (gymnotic transfection or gymnotic
delivery). The inhibitory effect of the modified oligonucleotides
on expression of TGF-beta2 mRNA, was determined 72 h after
treatment start. TGF-beta2 values were normalized to GAPDH and
oligonucleotide concentrations resulting in 50% reduction of
TGF-beta2 mRNA (=IC.sub.50 values) were calculated. Under gymnotic
delivery experimental conditions, the oligonucleotides enter the
cells and strongly inhibit the expression of TGF-beta2 mRNA. The
results of the experiments are shown in Table 3:
TABLE-US-00003 Name IC50 (.mu.M) ASPH_065 0.37 ASPH_071 0.371
ASPH_115 0.6 ASPH_069 0.655 ASPH_047 0.78 ASPH_080 0.81 ASPH_153
0.9 ASPH_045 1.21 ASPH_121 1.27 ASPH_036 1.5 ASPH_185 3.05 ASPH_003
3.62 ASPH_189 4.26
[0093] All the modified oligonucleotides show an IC.sub.50 in the
low micromolar or even submicromolar range, showing that they have
very high potency even without the requirement of a transfection
reagent.
Example 11
[0094] Human Panc-1 pancreatic cancer cells were treated with 10
.mu.M, 3.3 .mu.M, 1.1 .mu.M, 0.37 .mu.M, and 0.12 .mu.M of ASPH47,
ASPH190, ASPH191, ASPH192, and ASPH193, respectively, in the
absence of a transfecting agent (gymnotic transfection or gymntic
delivery). The inhibitory effect of the modified oligonucleotides
on expression of TGF-beta2 mRNA, was determined 72 h after
treatment start. TGF-beta2 values were normalized to GAPDH and
oligonucleotide concentrations resulting in 50% reduction of
TGF-beta2 mRNA (=IC.sub.50 values) were calculated. Under gymnotic
delivery experimental conditions, the oligonucleotides enter the
cells and strongly inhibit the expression of TGF-beta2 mRNA. The
results of the experiments are shown in Table 4:
TABLE-US-00004 Name IC50 (.mu.M) ASPH_047 0.76 ASPH_190 0.18
ASPH_191 0.97 ASPH_192 0.145 ASPH_193 0.144
[0095] All the modified oligonucleotides show an IC.sub.50 in the
submicromolar to lower submicromolar range, showing that they have
extremely high potency even without the requirement of a
transfection reagent.
Example 12
[0096] Human Panc-1 pancreatic cancer cells were transfected with
10 nM of ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003,
ASPH1004, ASPH1005, ASPH1006, ASPH 1007, ASPH1008, ASPH1009,
ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015,
ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021,
ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028,
ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034,
ASPH1035, ASPH1036, ASPH 1038, ASPH1039, ASPH1040, ASPH1041,
ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047,
ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1054,
ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, or
ASPH1061 and the control of SEQ ID NO. 145, respectively, in the
presence of a transfecting agent. The expression of TGF-beta1 mRNA
was determined 24 h after transfection. Significant reduction of
the expression of TGF-beta1 in Panc-1 cells is shown in FIG.
13.
Example 13
[0097] Mouse SMA-560 glioma cells were transfected with 10 nM of
ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005,
ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011,
ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017,
ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023,
ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030,
ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036,
ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042,
ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048,
ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054,
ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060,
ASPH1061, or ASPH1062 and the control of SEQ ID NO. 145,
respectively, in the presence of a transfecting agent. The
expression of TGF-beta1 mRNA was determined 24 h after
transfection. Significant reduction of the expression of TGF-beta1
in SMA-560 cells is shown in FIG. 14.
Example 14
[0098] In these experiments, human A172 glioma cells were
transfected with 10 nM of ASPH05, ASPH09, ASPH1000, ASPH1001,
ASPH1002, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008,
ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014,
ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020,
ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027,
ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033,
ASPH1034, ASPH1035, ASPH1036, ASPH1038, ASPH1039, ASPH1040,
ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046,
ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052,
ASPH1053, ASPH1054, ASPH1056, ASPH1057, ASPH1058, ASPH1059,
ASPH1060, ASPH1061, or ASPH1062, and the control of SEQ ID NO. 145,
respectively, in the presence of a transfecting agent. The
expression of TGF-beta1 and TGF-beta2 mRNA was determined 24 h
after transfection. Significant reduction of the expression of
TGF-beta1 mRNA is shown in FIG. 15. The dual TGF-beta1 and
TGF-beta2 reactive oligonucleotides ASPH05 shows a significant
reduction of the expression of both TGF-beta1 and TGF-beta2 mRNAs,
while the selective TGF-beta1 oligonucleotides significantly
inhibit TGF-beta1 mRNA expression.
Example 15
[0099] Human Panc-1 pancreatic cancer cells were treated with 3.3
.mu.M of ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1004,
ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011,
ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1017, ASPH1018,
ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1024, ASPH1026,
ASPH1027, ASPH1028, ASPH1029, ASPH1032, ASPH1033, ASPH1034,
ASPH1035, ASPH1036, ASPH1037, ASPH1038, ASPH1039, ASPH1040,
ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046,
ASPH1047, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053,
ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059,
ASPH1060, ASPH1061, or ASPH1062, or the control of SEQ ID NO. 145
in the absence of a transfecting agent (gymnotic transfection or
gymnotic delivery). The inhibitory effect of the modified
oligonucleotides on expression of TGF-beta1 and TGF-beta2 mRNA,
respectively, was determined 72 h after treatment start.
Significant reduction of the expression of TGF-beta1 mRNA is shown
in FIG. 16. The dual TGF-beta1 and TGF-beta2 reactive
oligonucleotides ASPH05 shows a significant reduction of the
expression of both TGF-beta1 and TGF-beta2 mRNAs, while the
selective TGF-beta1 oligonucleotides significantly inhibit
TGF-beta1 mRNA expression.
Example 16
[0100] Human A172 glioma cells were treated with 10 nM (in the
presence of a transfecting agent), of ASPH09, ASPH1047, ASPH1051,
ASPH1059, ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067,
ASPH1068, ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073,
ASPH1074, ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079,
ASPH1080, ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085,
ASPH1086, ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091,
ASPH1092, ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098,
ASPH1099, ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104,
ASPH1105, ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110,
ASPH1111, ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116,
ASPH1117, ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122,
ASPH1123, ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128,
ASPH1129, ASPH1130, ASPH1131, and ASPH1132, respective1, or the
positive control ASPH1047. The expression of TGF-beta1 (black
column), TGF-beta2 (white column) and TGF-beta3 (striped column)
mRNA was determined 24 h after transfection. Significant reduction
of the expression of TGF-beta1 mRNA is shown in FIG. 17. The
pan-specific TGF-beta1, TGF-beta2 and TGF-beta3 reactive
oligonucleotides ASPH0009, ASPH1096, ASPH1131, and ASPH1132 show a
significant reduction of the expression of all three isoforms,
while the selective TGF-beta1 oligonucleotides significantly
inhibit TGF-beta1 mRNA expression.
Example 17
[0101] Either human Panc-1 pancreatic cancer cells (FIG. 18a) or
mouse RenCa renal cell carcinoma cells (FIG. 18a) were treated with
3.3 .mu.M of ASPH09, ASPH1047, ASPH1051, ASPH1059, ASPH1063,
ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069,
ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075,
ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081,
ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087,
ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093,
ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100,
ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106,
ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112,
ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118,
ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124,
ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130,
ASPH1131, and ASPH1132, respectively, or the positive control
ASPH1047 in the absence of a transfecting agent (gymnotic
transfection or gymnotic delivery). The expression of TGF-beta1
(black column), TGF-beta2 (white column) and TGF-beta3 (striped
column) mRNA was determined 72 h after transfection. Significant
reduction of the expression of TGF-beta1 mRNA is shown in FIG. 17.
The pan-specific TGF-beta1, TGF-beta2 and TGF-beta3 reactive
oligonucleotides ASPH0009, ASPH1096, ASPH1131, and ASPH1132 show
significant reduction of the expression of all three isoforms,
while the selective TGF-beta1 oligonucleotides significantly
inhibit TGF-beta1 mRNA expression.
Example 18
[0102] Mice bearing human Panc-1 pancreatic carcinoma subcutaneous
tumors were treated with 1, 3, 10, and 30 mg/kg of ASPH47 under
various treatment schedules: Q1Dx1-d6 (single SC injection,
termination 5 days later), Q1Dx5-d6 (daily SC injection for 5 days,
termination 24 hours later), and Q1Dx5-d10 (daily SC injection for
5 days, termination 5 days later). There was a dose dependent
down-regulation of TGF-beta2 mRNA in the kidney of these animals.
TGF-beta2 down-regulation was persistent up to 5 days after the
last treatment with ASPH47, even after only single administration.
TGF-beta 2 expression was detected by bDNA assay (branched DNA
assay, which is a sandwich nucleic acid hybridization method that
uses bDNA molecules to amplify signal from captured target RNA) and
normalized to GAPDH. As shown in FIG. 22, data--representing
TGF-beta2 to GAPDH mRNA ratio--are shown as a box plot in which
median values and min. and max. values are presented (data
expressed as n=10, except n=9 for vehicle and 3 mg/kg Q1Dx1 d6
groups).
Example 19
[0103] Mice bearing human Panc-1 pancreatic carcinoma subcutaneous
tumors on both left and right flanks were treated with a daily
subcutaneous injection of 1, 5, 15 or 50 mg/kg oligonucleotides for
five consecutive days. The tumors were collected 24 hours after the
last treatment and snap frozen. TGF-beta mRNA expression in tumors
was detected by bDNA assay. Data--representing TGF-beta2 to GAPDH
mRNA ratio--are shown as a box plot in which median values and min.
and max. values are presented (data expressed as n=5). TGF-beta2
mRNA was down-regulated in tumors treated with various
oligonucleotides (FIG. 23). There was no significant TGF-beta1 mRNA
down-regulation in those groups (data not shown).
Example 20
[0104] Mice bearing human 786-0 renal cell carcinoma subcutaneous
tumors on both left and right flanks were treated with a daily
injection of 50 mg/kg oligonucleotides for five consecutive days.
The tumors were collected 24 hours after the last treatment and
snap frozen. TGF-beta mRNA expression in tumors was detected by
bDNA assay. There was significant down-regulation of TGF-beta2 mRNA
in tumors treated with ASPH05, ASPH17, ASPH26, ASPH36, ASPH45,
ASPH47, ASPH71, ASPH82, ASPH98, and ASPH105 (FIG. 24).
Data--representing TGF-beta2 to GAPDH mRNA ratio--are shown as a
box plot in which median values and min. and max. values are
presented (data expressed as n=10, except for ASPH71 group
n=9).
Example 21
[0105] Human Panc-1 pancreatic cancer cells were transfected with
20, 6.67, 2.22, 0.74, 0.25, 0.08 or 0.009 .mu.M of the modified
oligonucleotides ASPH47, ASPH1047, ASPH1106, ASPH1132, or ASPH47 in
combination with ASPH1047; results are shown in FIG. 26a to 26e).
Negative control is the scrambled oligonucleotide (scrLNA) of SEQ
ID No. 145 (FIG. 26 f). All cells were transfected in the absence
of transfecting agent (gymnotic transfection or gymnotic delivery).
The modified oligonucleotides were added to the cells for 3 days,
which were incubated at 37.degree. C. Thereafter medium was
exchanged with fresh oligonucleotide containing medium and cells
were incubated for further 4 days at 37.degree. C. TGF-beta1 and
TGF-beta2 protein levels in cell supernatants were determined by
ELISA. ASPH47 specifically inhibits the expression of TGF-beta2 in
a dose-dependent manner and does not have any target inhibiting
effect on TGF-beta1 (FIG. 26a). ASPH1047 specifically inhibits the
expression of TGF-beta1 and does not have any target inhibiting
effect on TGF-beta2 (FIG. 26b), or only a slight TGF-beta2
inhibiting effect at higher concentrations. Also ASPH1106 inhibits
TGF-beta1 expression in a dose dependent manner (FIG. 26c). The
pan-specific ASPH 1132 shows a dose-dependent inhibition of the
expression of TGF-beta1 and TGF-beta2 protein (FIG. 26d). When
ASPH47 and ASPH1047 are combined, the expression of both, TGF-beta1
and TGF-beta2 protein is inhibited in a dose dependent manner (FIG.
26e). The scrLNA of SEQ ID No. 145 does not show any inhibiting
effect on the expression of neither TGF-beta1 nor TGF-beta2, even
if the concentrations were doubled (40, 13.33, 4.44, 1.48, 0.49,
0.16, 0.05, or 0.02 .mu.M) in comparison to the individual
concentrations of ASPH47, ASPH1047, ASPH1106, or ASPH1132. Results
for TGF-beta1 are indicated in diamonds, and results for TGF-beta2
in squares in FIGS. 26a to 26f.
Example 22
[0106] Human Panc-1 pancreatic cancer cells (FIG. 27a) or mouse
RenCa renal cell carcinoma cells (FIG. 27b) were treated with 3.3
.mu.M of ASPH0009, ASPH1132, ASPH2000, ASPH2001, ASPH2002,
ASPH2003, ASPH2004, ASPH2005, ASPH2006, ASPH2007, ASPH2009,
ASPH2010, ASPH2012, ASPH2013, ASPH2014 ASPH2015, ASPH2016,
ASPH2017, ASPH2018, ASPH2019, ASPH2020, ASPH2021, ASPH2023,
ASPH2024, ASPH2025, ASPH23026, ASPH2027, ASPH2028, ASPH2029,
ASPH2030, ASPH2031, ASPH2032, ASPH2033, ASPH2034, ASPH2035,
ASPH2036, ASPH2037, ASPH2038, ASPH2039, ASPH2040, ASPH2041,
ASPH2043, ASPH2044, ASPH2045, ASPH2046, ASPH2047, ASPH2048,
ASPH2049, ASPH2050, ASPH2052, ASPH2053, ASPH2054, ASPH2055,
ASPH2056, ASPH2057, ASPH2060, ASPH2061, ASPH2062, ASPH2063,
ASPH2064, ASPH2065, or ASPH2066 in the absence of a transfecting
agent (gymnotic transfection or gymnotic delivery). The expression
of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3
(striped column) mRNA was determined 72 h after transfection.
Significant reduction of the expression of TGF-beta3 mRNA is shown
in FIGS. 27a and 27b. As anticipated from the sequences, the
TGF-beta1, -beta2 and -beta3 reactive oligonucleotide
ASPH.sub.--0009 (pan-selective) and ASPH.sub.--1132 that has 100%
homology to mRNAs of human TGF-beta1 and -beta3 but has a mismatch
to TGF-beta2 show significant reduction of the expression of all
three isoforms. The selective TGF-beta3 oligonucleotides only
significantly inhibit TGF-beta3 mRNA expression.
Example 23
[0107] Human A172 glioma cells were treated for 24 h with 10 nM (in
the presence of a transfecting agent), of ASPH0009, ASPH1132,
ASPH2000, ASPH2001, ASPH2002, ASPH2003, ASPH2004, ASPH2006,
ASPH2007, ASPH2008, ASPH2009, ASPH2010, ASPH2011, ASPH2012,
ASPH2013, ASPH2014, ASPH2016, ASPH2017, ASPH2018, ASPH2020,
ASPH2021, ASPH2022, ASPH2023, ASPH2024, ASPH2025, ASPH2026,
ASPH2027, ASPH2028, ASPH2029, ASPH2030, ASPH2031, ASPH2032,
ASPH2033, ASPH2034, ASPH2035, ASPH2036, ASPH2037, ASPH2038,
ASPH2039, ASPH2040, ASPH2041, ASPH2042, ASPH2043, ASPH2044,
ASPH2045, ASPH2047, ASPH2049, ASPH2050, ASPH2051, ASPH2052,
ASPH2053, ASPH2054, ASPH2056, ASPH2057, ASPH2058, ASPH2059,
ASPH2060, ASPH2061, ASPH2062, ASPH2063, or ASPH2066. The expression
of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3
(striped column) mRNA was then determined from cell extracts by
bDNA assay. Significant reduction of the expression of TGF-beta3
mRNA is shown in FIG. 28. As anticipated from the sequences, the
TGF-beta1, -beta2 and -beta3 reactive oligonucleotide)
ASPH.sub.--0009 (pan-selective) and ASPH.sub.--1132 that has 100%
homology to mRNAs of human TGF-beta1 and -beta3 but has a mismatch
to TGF-beta2 show significant reduction of the expression of all
three isoforms. The selective TGF-beta3 oligonucleotides only
significantly inhibit TGF-beta3 mRNA expression.
Example 24
Target mRNA Downregulation in Rabbit Cells
[0108] Sequences of selected oligonucleotides were aligned with
rabbit mRNA sequences of TGF-beta1 and 2. ASPH.sub.--0036
(TGF-beta2 selective antisense oligonucleotide, based on human mRNA
sequence) showed 100% homology with rabbit TGF-beta2 mRNA, while
ASPH.sub.--1059 (TGF-beta1 selective antisense oligonucleotide,
based on human mRNA sequence) showed 100% homology with rabbit
TGF-beta1 mRNA.
[0109] Rabbit Rab-9 skin fibroblasts were treated with 5 nM or 20
nM of either ASPH.sub.--0036 and ASPH.sub.--1059 in the presence of
a transfecting agent for 24 hr. The expression of TGF-beta1 and
TGF-beta2 mRNA was then determined in cell extracts by bDNA assay.
Significant reduction of the expression of TGF-beta1 mRNA (51 and
77% at 5 and 20 nM, respectively) was achieved with
ASPH.sub.--1059. Significant reduction of TGF-beta2 mRNA (79 and
80% at 5 and 20 nM, respectively) was achieved with
ASPH.sub.--0036.
Example 25
Tissue Biodistribution and Target mRNA Downregulation Following
Systemic Administration of ASPH.sub.--0047 in Balb/c Mice
[0110] Balb/C mice were treated with a single subcutaneous
injection of ASPH.sub.--0047 (formulated in sterile physiological
saline) at 5, 20 and 50 mg/kg animal body weight. Plasma and
tissues were collected at the indicated times (from 3 individual
animals), immediately snap-frozen and stored at -80.degree. C.
until analysis with an AEX-HPLC method (plasma/tissue PK) or for
measurement of TGF-.beta.2 and GAPDH mRNA levels by bDNA assay.
TGF-.beta.2 mRNA levels were expressed relative to GAPDH mRNA
expression level in corresponding samples.
[0111] The data depict that a single subcutaneous bolus
administration of 50 mg/kg ASPH.sub.--0047 resulted in rapid
transfer of the drug from subcutaneous to circulating blood
compartments (T.sub.MAX of .about.5-30 min), biphasic
pharmacokinetic profile in plasma, with rapid initial elimination
phase (within the first 24 hrs), followed by long terminal
half-life (FIG. 29a). It is further demonstrated that a marked
long-lasting accumulation of the drug in various selected tissues.
The major target organ (highest exposure/C.sub.MAX) is the kidney,
then the liver, skin and spleen, and lowest in the brain (data not
shown). As also depicted in FIG. 29b, ASPH.sub.--0047 remained in
the kidney tissue with pharmacological relevant doses (.about.50
.mu.g/gr, equivalent to 10 .mu.M) from 24 h and for up to 14 days,
with consequent long-lasting and marked suppression of TGF-.beta.2
mRNA expression in the kidney tissue, with effective .about.80%
target mRNA downregulation observed for at least 14 days.
Example 26
[0112] Immunodeficient mice were injected subcutaneously with human
786-O renal cell carcinoma cells (FIG. 30A), pancreatic Panc1
cancer cells (FIG. 30B, C), or mouse SMA-560 glioma cells (FIG.
30D). When subcutaneous tumors reached the size of 100-300 mm.sup.3
(established tumors), animals were treated subcutaneously, Q1Dx5,
with saline (Mock), control oligonucleotide (Control; 50 mg/kg),
inactive oligonucleotides in this context (e.g., ASPH.sub.--0065
and ASPH.sub.--0071; 50 mg/kg) or ASPH.sub.--0047 at 50 mg/kg, or
the indicated doses. Tumors (FIG. 30A-D) and kidneys (FIG. 30E-F)
were collected 24 hr after the last administration. Tumors/kidneys
were then further processed for determination of TGF-.quadrature.2
and GAPDH mRNA levels by bDNA assay. In these experiments, control
oligonucleotide was a 18-mer, 3+3 LNA gapmer scrambled sequence.
Results are expressed as TGF-beta2/GAPDH mRNA ratio, and each
individual tested sample is represented with median values
indicated as red line. Under described experimental conditions
(schedule and route of administration), systemic repeated
administrations of ASPH.sub.--0047 in Balb/c mice led to a
sequence-specific downregulation of TGF-beta 2 mRNA in established
subcutaneous tumors and kidneys.
Example 27
[0113] Balb/c mice were injected with mouse Renca cells into renal
subcapsule (FIG. 31A, B) or i.v. (FIG. 31C, D) on Day 0. Systemic
treatment with vehicle or indicated oligonucleotides started on Day
7 (FIG. 31A; 50 mg/kg, s.c., twice weekly), on Day 1 (FIG. 31B;
12.5 mg/kg, s.c., twice weekly) for two consecutive weeks, or on
Day 7 (FIGS. 31C and 31D; indicated doses, s.c., twice weekly) for
26-27 days. Number of lung metastasis was macroscopically
evaluated, and level of lung metastasis was determined by either
number of metastasis (FIG. 31A, C) or based on lung weight (FIG.
31B, D). Results are represented as box plot; with median values,
upper and lower quartiles, and 90th and 10th percentiles. Under
described experimental designs, Balb/c mice treated with
ASPH.sub.--0047 showed a reduced number of lung metastasis or
reduced lung weight (lung weight correlates to extent of lung
metastasis) in mouse Renca RCC models.
Example 28
[0114] Human Panc-1 pancreatic cancer cells were treated with 3.3
.mu.M of the indicated oligonucleotides in the absence of
transfecting agent (gymnotic transfection or gymnotic delivery).
The expression of TGF-beta1 (black column), TGF-beta2 (white
column) and TGF-beta3 (striped column) mRNA was determined 72 h
after transfection. Significant reduction of the expression of
TGF-beta1 mRNA is shown in FIG. 32. The selective TGF-beta1
oligonucleotides only significantly inhibit TGF-beta1 mRNA
expression while the control oligonucleotide LNA-scr does not
affect expression of any TGF-beta isoform.
Example 29
[0115] Balb/c mice were injected with mouse 4T1 cells into mammary
fat pad on Day 0. Systemic treatment with saline (Mock),
pan-TGF-beta antibody (1D11), control oligonucleotide (LNA-scr), or
ASPH.sub.--0047 started on Day 3 (30 mg/kg, s.c., twice weekly) and
continued until D28, when animals were sacrificed. Number of lung
metastasis was macroscopically evaluated, and level of lung
metastasis was determined by either number of metastasis (left
panel) or based on lung weight (right panel). Under described
experimental design, treatment with ASPH.sub.--0047 reduced
metastasis to the lungs, whereas the positive control, monoclonal
TGF-beta antibody 1D11 had no effect on pulmonary metastasis in
this model.
Example 30
[0116] CB17 SCID or Balb/c nude mice (n=3-5, except ASPH.sub.--0018
n=1 and ASPH.sub.--0037 n=2) were treated with 14-15 mg/kg of
indicated LNA-modified oligonucleotides for four or five
consecutive days (Q1Dx4-5). Plasma was collected 24 h after the
last treatment and ALT levels were determined in plasma. Results
are expressed as median values. Under this experimental condition,
only 6/48 (12.5%) of tested oligonucleotides induced marked
increase in plasma ALT (>300 units/l) indicating liver toxicity.
The following Table 7 shows liver toxicity of systemically
administered LNA-modified oligonucleotides:
TABLE-US-00005 Name ALT (units/l) ASPH_0001 20.5 ASPH_0003 20.0
ASPH_0005 33.0 ASPH_0009 834.0 ASPH_0017 55.0 ASPH_0018 7723.0
ASPH_0022 28.5 ASPH_0026 77.0 ASPH_0027 75.0 ASPH_0035 25.0
ASPH_0036 131.5 ASPH_0037 161.0 ASPH_0041 655.0 ASPH_0045 27.5
ASPH_0046 3199.0 ASPH_0047 42.5 ASPH_0048 29.5 ASPH_0065 27.0
ASPH_0069 32.5 ASPH_0071 23.5 ASPH_0080 34.0 ASPH_0082 31.0
ASPH_0098 33.0 ASPH_0105 40.0 ASPH_0115 985.5 ASPH_0190 902.0
ASPH_0191 36.5 ASPH_0192 49.5 ASPH_0193 35.0 ASPH_0005_C1 25.5
ASPH_0005_C2 35.5 ASPH_0005_C3 25.0 ASPH_0036_C1 34.0 ASPH_0036_C2
26.0 ASPH_0036_C3 39.0 ASPH_0045_C1 38.5 ASPH_0045_C2 23.5
ASPH_0045_C3 65.0 ASPH_0047_C1 35.5 ASPH_0047_C2 30.0 ASPH_0047_C3
29.5 ASPH_0047_C4 52.5 ASPH_0047_C5 28.0 ASPH_0047_C6 33.5
ASPH_0047_C7 37.0 ASPH_0047_C8 32.0 ASPH_0047_C9 49.0 ASPH_0047_C10
32.5
Sequence CWU 1
1
40215882DNAHomo sapiens 1gtgatgttat ctgctggcag cagaaggttc
gctccgagcg gagctccaga agctcctgac 60aagagaaaga cagattgaga tagagataga
aagagaaaga gagaaagaga cagcagagcg 120agagcgcaag tgaaagaggc
aggggagggg gatggagaat attagcctga cggtctaggg 180agtcatccag
gaacaaactg aggggctgcc cggctgcaga caggaggaga cagagaggat
240ctattttagg gtggcaagtg cctacctacc ctaagcgagc aattccacgt
tggggagaag 300ccagcagagg ttgggaaagg gtgggagtcc aagggagccc
ctgcgcaacc ccctcaggaa 360taaaactccc cagccagggt gtcgcaaggg
ctgccgttgt gatccgcagg gggtgaacgc 420aaccgcgacg gctgatcgtc
tgtggctggg ttggcgtttg gagcaagaga aggaggagca 480ggagaaggag
ggagctggag gctggaagcg tttgcaagcg gcggcggcag caacgtggag
540taaccaagcg ggtcagcgcg cgcccgccag ggtgtaggcc acggagcgca
gctcccagag 600caggatccgc gccgcctcag cagcctctgc ggcccctgcg
gcacccgacc gagtaccgag 660cgccctgcga agcgcaccct cctccccgcg
gtgcgctggg ctcgccccca gcgcgcgcac 720acgcacacac acacacacac
acacacacgc acgcacacac gtgtgcgctt ctctgctccg 780gagctgctgc
tgctcctgct ctcagcgccg cagtggaagg caggaccgaa ccgctccttc
840tttaaatata taaatttcag cccaggtcag cctcggcggc ccccctcacc
gcgctcccgg 900cgcccctccc gtcagttcgc cagctgccag ccccgggacc
ttttcatctc ttcccttttg 960gccggaggag ccgagttcag atccgccact
ccgcacccga gactgacaca ctgaactcca 1020cttcctcctc ttaaatttat
ttctacttaa tagccactcg tctctttttt tccccatctc 1080attgctccaa
gaattttttt cttcttactc gccaaagtca gggttccctc tgcccgtccc
1140gtattaatat ttccactttt ggaactactg gccttttctt tttaaaggaa
ttcaagcagg 1200atacgttttt ctgttgggca ttgactagat tgtttgcaaa
agtttcgcat caaaaacaac 1260aacaacaaaa aaccaaacaa ctctccttga
tctatacttt gagaattgtt gatttctttt 1320ttttattctg acttttaaaa
acaacttttt tttccacttt tttaaaaaat gcactactgt 1380gtgctgagcg
cttttctgat cctgcatctg gtcacggtcg cgctcagcct gtctacctgc
1440agcacactcg atatggacca gttcatgcgc aagaggatcg aggcgatccg
cgggcagatc 1500ctgagcaagc tgaagctcac cagtccccca gaagactatc
ctgagcccga ggaagtcccc 1560ccggaggtga tttccatcta caacagcacc
agggacttgc tccaggagaa ggcgagccgg 1620agggcggccg cctgcgagcg
cgagaggagc gacgaagagt actacgccaa ggaggtttac 1680aaaatagaca
tgccgccctt cttcccctcc gaaaatgcca tcccgcccac tttctacaga
1740ccctacttca gaattgttcg atttgacgtc tcagcaatgg agaagaatgc
ttccaatttg 1800gtgaaagcag agttcagagt ctttcgtttg cagaacccaa
aagccagagt gcctgaacaa 1860cggattgagc tatatcagat tctcaagtcc
aaagatttaa catctccaac ccagcgctac 1920atcgacagca aagttgtgaa
aacaagagca gaaggcgaat ggctctcctt cgatgtaact 1980gatgctgttc
atgaatggct tcaccataaa gacaggaacc tgggatttaa aataagctta
2040cactgtccct gctgcacttt tgtaccatct aataattaca tcatcccaaa
taaaagtgaa 2100gaactagaag caagatttgc aggtattgat ggcacctcca
catataccag tggtgatcag 2160aaaactataa agtccactag gaaaaaaaac
agtgggaaga ccccacatct cctgctaatg 2220ttattgccct cctacagact
tgagtcacaa cagaccaacc ggcggaagaa gcgtgctttg 2280gatgcggcct
attgctttag aaatgtgcag gataattgct gcctacgtcc actttacatt
2340gatttcaaga gggatctagg gtggaaatgg atacacgaac ccaaagggta
caatgccaac 2400ttctgtgctg gagcatgccc gtatttatgg agttcagaca
ctcagcacag cagggtcctg 2460agcttatata ataccataaa tccagaagca
tctgcttctc cttgctgcgt gtcccaagat 2520ttagaacctc taaccattct
ctactacatt ggcaaaacac ccaagattga acagctttct 2580aatatgattg
taaagtcttg caaatgcagc taaaattctt ggaaaagtgg caagaccaaa
2640atgacaatga tgatgataat gatgatgacg acgacaacga tgatgcttgt
aacaagaaaa 2700cataagagag ccttggttca tcagtgttaa aaaatttttg
aaaaggcggt actagttcag 2760acactttgga agtttgtgtt ctgtttgtta
aaactggcat ctgacacaaa aaaagttgaa 2820ggccttattc tacatttcac
ctactttgta agtgagagag acaagaagca aatttttttt 2880aaagaaaaaa
ataaacactg gaagaattta ttagtgttaa ttatgtgaac aacgacaaca
2940acaacaacaa caacaaacag gaaaatccca ttaagtggag ttgctgtacg
taccgttcct 3000atcccgcgcc tcacttgatt tttctgtatt gctatgcaat
aggcaccctt cccattctta 3060ctcttagagt taacagtgag ttatttattg
tgtgttacta tataatgaac gtttcattgc 3120ccttggaaaa taaaacaggt
gtataaagtg gagaccaaat actttgccag aaactcatgg 3180atggcttaag
gaacttgaac tcaaacgagc cagaaaaaaa gaggtcatat taatgggatg
3240aaaacccaag tgagttatta tatgaccgag aaagtctgca ttaagataaa
gaccctgaaa 3300acacatgtta tgtatcagct gcctaaggaa gcttcttgta
aggtccaaaa actaaaaaga 3360ctgttaataa aagaaacttt cagtcagaat
aagtctgtaa gttttttttt ttctttttaa 3420ttgtaaatgg ttctttgtca
gtttagtaaa ccagtgaaat gttgaaatgt tttgacatgt 3480actggtcaaa
cttcagacct taaaatattg ctgtatagct atgctatagg ttttttcctt
3540tgttttggta tatgtaacca tacctatatt attaaaatag atggatatag
aagccagcat 3600aattgaaaac acatctgcag atctcttttg caaactatta
aatcaaaaca ttaactactt 3660tatgtgtaat gtgtaaattt ttaccatatt
ttttatattc tgtaataatg tcaactatga 3720tttagattga cttaaatttg
ggctcttttt aatgatcact cacaaatgta tgtttctttt 3780agctggccag
tacttttgag taaagcccct atagtttgac ttgcactaca aatgcatttt
3840ttttttaata acatttgccc tacttgtgct ttgtgtttct ttcattatta
tgacataagc 3900tacctgggtc cacttgtctt ttcttttttt tgtttcacag
aaaagatggg ttcgagttca 3960gtggtcttca tcttccaagc atcattacta
accaagtcag acgttaacaa atttttatgt 4020taggaaaagg aggaatgtta
tagatacata gaaaattgaa gtaaaatgtt ttcattttag 4080caaggattta
gggttctaac taaaactcag aatctttatt gagttaagaa aagtttctct
4140accttggttt aatcaatatt tttgtaaaat cctattgtta ttacaaagag
gacacttcat 4200aggaaacatc tttttcttta gtcaggtttt taatattcag
ggggaaattg aaagatatat 4260attttagtcg atttttcaaa aggggaaaaa
agtccaggtc agcataagtc attttgtgta 4320tttcactgaa gttataaggt
ttttataaat gttctttgaa ggggaaaagg cacaagccaa 4380tttttcctat
gatcaaaaaa ttctttcttt cctctgagtg agagttatct atatctgagg
4440ctaaagttta ccttgcttta ataaataatt tgccacatca ttgcagaaga
ggtatcctca 4500tgctggggtt aatagaatat gtcagtttat cacttgtcgc
ttatttagct ttaaaataaa 4560aattaatagg caaagcaatg gaatatttgc
agtttcacct aaagagcagc ataaggaggc 4620gggaatccaa agtgaagttg
tttgatatgg tctacttctt ttttggaatt tcctgaccat 4680taattaaaga
attggatttg caagtttgaa aactggaaaa gcaagagatg ggatgccata
4740atagtaaaca gcccttgtgt tggatgtaac ccaatcccag atttgagtgt
gtgttgatta 4800tttttttgtc ttccactttt ctattatgtg taaatcactt
ttatttctgc agacattttc 4860ctctcagata ggatgacatt ttgttttgta
ttattttgtc tttcctcatg aatgcactga 4920taatatttta aatgctctat
tttaagatct cttgaatctg tttttttttt ttttaatttg 4980ggggttctgt
aaggtcttta tttcccataa gtaaatattg ccatgggagg ggggtggagg
5040tggcaaggaa ggggtgaagt gctagtatgc aagtgggcag caattatttt
tgtgttaatc 5100agcagtacaa tttgatcgtt ggcatggtta aaaaatggaa
tataagatta gctgttttgt 5160attttgatga ccaattacgc tgtattttaa
cacgatgtat gtctgttttt gtggtgctct 5220agtggtaaat aaattatttc
gatgatatgt ggatgtcttt ttcctatcag taccatcatc 5280gagtctagaa
aacacctgtg atgcaataag actatctcaa gctggaaaag tcataccacc
5340tttccgattg ccctctgtgc tttctccctt aaggacagtc acttcagaag
tcatgcttta 5400aagcacaaga gtcaggccat atccatcaag gatagaagaa
atccctgtgc cgtcttttta 5460ttcccttatt tattgctatt tggtaattgt
ttgagattta gtttccatcc agcttgactg 5520ccgaccagaa aaaatgcaga
gagatgtttg caccatgctt tggctttctg gttctatgtt 5580ctgccaacgc
cagggccaaa agaactggtc tagacagtat cccctgtagc cccataactt
5640ggatagttgc tgagccagcc agatataaca agagccacgt gctttctggg
gttggttgtt 5700tgggatcagc tacttgcctg tcagtttcac tggtaccact
gcaccacaaa caaaaaaacc 5760caccctattt cctccaattt ttttggctgc
tacctacaag accagactcc tcaaacgagt 5820tgccaatctc ttaataaata
ggattaataa aaaaagtaat tgtgactcaa aaaaaaaaaa 5880aa
5882214DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
2gaccagatgc agga 14316DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 3gcgaccgtga ccagat 16414DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 4gcgcgaccgt gacc
14513DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
5agcgcgaccg tga 13614DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 6gaccgtgacc agat 14712DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 7ctgcccgcgg at
12813DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
8tctgcccgcg gat 13915DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 9ggatctgccc gcgga 151017DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 10cttgctcagg atctgcc
171120DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
11gctcaggatc tgcccgcgga 201213DNAArtificial Sequencemodified
TGF-beta2 oligonucleotide 12ggatcgcctc gat 131312DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 13ccgcggatcg cc
121417DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
14acctccttgg cgtagta 171516DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 15cctccttggc gtagta 161615DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 16ctccttggcg tagta
151714DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
17tccttggcgt agta 141813DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 18cagaagttgg cat 131913DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 19aagtgggcgg gat
132012DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
20gcgggatggc at 122113DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 21gaaatcacct ccg 132213DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 22aagtgggcgg gat
132313DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
23tgtagcgctg ggt 132413DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 24cgaaggagag cca 132514DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 25tcgcgctcgc aggc
142614DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
26aagtgggcgg gatg 142714DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 27atgtagcgct gggt 142814DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 28cgaaggagag ccat
142915DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
29gaaagtgggc gggat 153015DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 30cgaaggagag ccatt 153116DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 31cgatcctctt gcgcat
163216DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
32aagtgggcgg gatggc 163317DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 33gatggaaatc acctccg 173417DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 34aaacctcctt ggcgtag
173517DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
35tagaaagtgg gcgggat 173613DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 36ggcgggatgg cat 133716DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 37gggtctgtag aaagtg
163815DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
38gaaggagagc cattc 153915DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 39ccaggttcct gtctt 154015DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 40tctgatcacc actgg
154117DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
41tttctgatca ccactgg 174215DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 42gtctgtagga gggca 154316DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 43agtctgtagg agggca
164413DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
44tctgtaggag ggc 134517DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 45cagatgccag ttttaac 174617DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 46caaagtattt ggtctcc
174717DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
47ccttaagcca tccatga 174815DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 48gtactggcca gctaa 154920DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 49gcctcgatcc tcttgcgcat
205020DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
50aaacctcctt ggcgtagtac 205120DNAArtificial Sequencemodified
TGF-beta2 oligonucleotide 51gaaagtgggc gggatggcat
205217DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
52gaattgctcg cttaggg 175317DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 53cgtcgcggtt gcgttca 175417DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 54cgtggcctac accctgg
175517DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
55ttctaaagca ataggcc 175617DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 56agaatggtta gaggttc 175717DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 57tctgaactag taccgcc
175817DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
58cccattaata tgacctc 175917DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 59tttagttaga accctaa 176017DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 60cctcagatat agataac
176117DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
61tactattatg gcatccc 176217DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 62tgcccacttg catacta 176317DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 63agcgtaattg gtcatca
176417DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
64cgttggcaga acataga 176517DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 65gggatactgt ctagacc 176617DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 66attggcaact cgtttga
176716DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
67cgtcaggcta atattc 166816DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 68ggatgactcc ctagac 166916DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 69gtcgcggttg cgttca
167016DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
70ctcggtactc ggtcgg 167116DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 71ggttcggtcc tgcctt 167216DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 72aataggccgc atccaa
167316DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
73aactagtacc gccttt 167416DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 74tcggtcatat aataac 167515DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 75agaccgtcag gctaa
157615DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
76gtcgcggttg cgttc 157715DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 77ttccactgcg gcgct
157815DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
78aaggagcggt tcggt 157915DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 79ctcgggtgcg gagtg 158015DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 80ctgactttgg cgagt
158115DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
81gataggaacg gtacg 158215DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 82cactttggat tcccg 158314DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 83gtcgcggttg cgtt
148414DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
84tacaccctgg cggg 148514DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 85ctcggtactc ggtc 148614DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 86aggagcggtt cggt
148714DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
87gtctcgggtg cgga 148814DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 88tacgggacgg gcag 148914DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 89cgtcgctcct ctcg
149014DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
90tagcgctggg ttgg 149114DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 91aagcaatagg ccgc 149214DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 92tacgggcatg ctcc
149314DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
93aggcgcggga tagg 149414DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 94tttggattcc cgcc 149514DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 95accactagag cacc
149614DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
96gcgttggcag aaca 149713DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 97ttgctcgctt agg 139813DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 98gtcgcggttg cgt
139913DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
99ggcgctcggt act 1310013DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 100atctgaactc ggc 1310113DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 101cggttggtct gtt
1310213DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
102tccaccctag atc 1310313DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 103ctagtaccgc ctt 1310413DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 104ggtcggcagt caa
1310512DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
105cttgcgacac cc 1210612DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 106gagcggttcg gt 1210714DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 107acacagtagt gcat
1410814DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
108gggtctgtag aaag 1410914DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 109ggttggagat gtta 1411014DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 110tgggttggag atgt
1411114DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
111gctgggttgg agat 1411214DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 112gcgctgggtt ggag 1411314DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 113agcgctgggt tgga
1411414DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
114tagcgctggg ttgg 1411514DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 115gtagcgctgg gttg 1411614DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 116gatgtagcgc tggg
1411714DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
117ccattcgcct tctg 1411814DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 118gagagccatt cgcc 1411914DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 119agcagggaca gtgt
1412014DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
120gcaggagatg tggg 1412114DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 121cggttggtct gttg 1412214DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 122ccggttggtc tgtt
1412314DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
123gccggttggt ctgt 1412414DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 124agttggcatt gtac 1412514DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 125ggttagaggt tcta
1412614DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
126atggttagag gttc 1412714DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 127agaatggtta gagg 1412814DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 128agagaatggt taga
1412914DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
129cgttgtcgtc gtca 1413014DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 130accaaggctc tctt 1413114DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 131gcttcttgtc tctc
1413214DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
132ggaacggtac gtac 1413314DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 133taggaacggt acgt 1413414DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 134gggataggaa cggt
1413514DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
135cgcgggatag gaac 1413614DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 136aggcgcggga tagg 1413714DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 137gtcaagctgg atgg
1413813DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
138tctgtaggag ggc 1313914DNAArtificial Sequencemodified TGF-beta2
oligonucleotide 139gaccagatgc agga 1414015DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 140ctccttggcg tagta
1514116DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
141cctccttggc gtagta 1614217DNAArtificial Sequencemodified
TGF-beta2 oligonucleotide 142cagatgccag ttttaac
1714317DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
143agcgtaattg gtcatca 1714417DNAArtificial Sequencefake sequence
144aaaaaaaaaa aaaaaaa 1714518DNAArtificial Sequencefake sequence
145aaaaaaaaaa aaaaaaaa 1814614DNAArtificial Sequencemodified
TGF-beta2 oligonucleotide 146agtatttggt ctcc 1414714DNAArtificial
Sequencemodified TGF-beta2 oligonucleotide 147aagtatttgg tctc
1414817DNAArtificial Sequencemodified TGF-beta2 oligonucleotide
148caaagtattt ggtctcc 1714915DNAArtificial Sequencemodified
TGF-beta2 oligonucleotide 149aagtatttgg tctcc 1515017DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 150agctcgtccc tcctccc
1715117DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
151gagggctggt ccggaat 1715217DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 152cgagggctgg tccggaa
1715317DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
153gagggcggca tggggga 1715417DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 154gcgggtgctg ttgtaca
1715517DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
155cgcgggtgct gttgtac 1715617DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 156gtcgcgggtg ctgttgt
1715717DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
157ggtcgcgggt gctgttg 1715817DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 158ccggtcgcgg gtgctgt
1715917DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
159cccggtcgcg ggtgctg 1716017DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 160agcacgcggg tgacctc
1716117DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
161ttagcacgcg ggtgacc 1716217DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 162gggctcgtgg atccact
1716317DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
163ccttgggctc gtggatc 1716417DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 164tggcatggta gcccttg
1716516DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
165cgagggctgg tccgga 1616616DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 166gcgggtgctg ttgtac 1616716DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 167gcacgcgggt gacctc
1616816DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
168ccttgggctc gtggat 1616916DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 169ggcatggtag cccttg 1617014DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 170gggtgctgtt gtac
1417114DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
171tcgcgggtgc tgtt 1417214DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 172gtcgcgggtg ctgt 1417314DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 173ctcgtggatc cact
1417414DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
174atggtagccc ttgg 1417514DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 175tggcatggta gccc 1417614DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 176gaagttggca tggt
1417713DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
177tcgcgggtgc tgt 1317813DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 178cacccggtcg cgg 1317913DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 179ccacccggtc gcg
1318013DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
180cgccaggaat tgt 1318113DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 181ggctcgtgga tcc 1318213DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 182tgggctcgtg gat
1318313DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
183gcatggtagc cct 1318413DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 184agttggcatg gta 1318513DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 185ttgcaggagc gca
1318617DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
186attagcacgc gggtgac 1718717DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 187accattagca cgcgggt
1718817DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
188caccattagc acgcggg 1718917DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 189ccaccattag cacgcgg
1719017DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
190tccaccatta gcacgcg 1719117DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 191tccaccttgg gcttgcg
1719216DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
192ttagcacgcg ggtgac 1619316DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 193accattagca cgcggg 1619416DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 194caccattagc acgcgg
1619515DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
195caccattagc acgcg 1519615DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 196gcggcacgca gcacg 1519714DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 197tcgatgcgct tccg
1419814DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
198tagcacgcgg gtga 1419914DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 199attagcacgc gggt 1420014DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 200cattagcacg cggg
1420114DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
201accattagca cgcg 1420214DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 202caccattagc acgc 1420314DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 203ccaccattag cacg
1420414DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
204tccaccatta gcac 1420514DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 205gaccttgctg tact 1420614DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 206ggaccttgct gtac
1420714DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
207aggaccttgc tgta 1420814DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 208cggcacgcag cacg 1420914DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 209accttgggct tgcg
1421013DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
210ttagcacgcg ggt 1321113DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 211accattagca cgc 1321213DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 212cggcacgcag cac
1321317DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
213caccagctcc atgtcga 1721417DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 214tcgcgggtgc tgttgta
1721517DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
215gtgtccaggc tccaaat 1721616DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 216gctcgtccct cctccc 1621716DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 217accagctcgt ccctcc
1621816DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
218ggaggccccg cccctg 1621916DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 219catgggggag gcggcg 1622016DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 220accagctcca tgtcga
1622116DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
221ggtcgcgggt gctgtt 1622216DNAArtificial Sequencemodified
TGF-beta1 oligonucleotide 222ggaccttgct gtactg 1622316DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 223tccaccttgg gcttgc
1622415DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
224agctcgtccc tcctc 1522515DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 225ccagctcgtc cctcc 1522615DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 226gagggctggt ccgga
1522715DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
227tcccgagggc tggtc 1522815DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 228cggcatgggg gaggc 1522915DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 229cagctccatg tcgat
1523015DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
230accagctcca tgtcg 1523115DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 231tcgcgggtgc tgttg 1523215DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 232gtcgcgggtg ctgtt
1523315DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
233ggtcgcgggt gctgt 1523415DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 234agcacgcggg tgacc 1523515DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 235tagcacgcgg gtgac
1523615DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
236cattagcacg cgggt 1523715DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 237tccaccatta gcacg 1523815DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 238ccaggaattg ttgct
1523915DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
239ttgggctcgt ggatc 1524015DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 240cttgggctcg tggat 1524115DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 241ttggcatggt agccc
1524215DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
242gaagttggca tggta 1524315DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 243agaagttggc atggt 1524415DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 244tgtccaggct ccaaa
1524515DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
245aggaccttgc tgtac 1524615DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 246caccttgggc ttgcg 1524714DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 247agctcgtccc tcct
1424814DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
248cagctcgtcc ctcc 1424914DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 249accagctcgt ccct 1425014DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 250cccgagggct ggtc
1425114DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
251gcggcatggg ggag 1425214DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 252gtcttgcagg tgga 1425314DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 253tcgatgcgct tccg
1425414DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
254ggacaggatc tggc 1425514DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 255acctccccct ggct 1425614DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 256accattagca cgcg
1425714DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
257cagcagttct tctc 1425814DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 258tacagctgcc gcac 1425914DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 259agttggcatg gtag
1426014DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
260aagttggcat ggta 1426114DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 261gaagttggca tggt 1426214DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 262tccaggctcc aaat
1426314DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
263accttgctgt actg 1426414DNAArtificial Sequencemodified TGF-beta1
oligonucleotide 264ttgcaggagc gcac 1426514DNAArtificial
Sequencemodified TGF-beta1 oligonucleotide 265gcagaagttg gcat
1426614DNAArtificial Sequencemodified TGF-beta1 oligonucleotide
266cgggtgctgt tgta 1426717DNAArtificial Sequencemodified TGF-beta
oligonucleotide 267cccagcggca acggaaa 1726817DNAArtificial
Sequencemodified TGF-beta oligonucleotide 268caagaggtcc ccgcgcc
1726917DNAArtificial Sequencemodified TGF-beta oligonucleotide
269gcgtccccgg cggcaaa 1727017DNAArtificial Sequencemodified
TGF-beta oligonucleotide 270ggtcggcgac tcccgag 1727117DNAArtificial
Sequencemodified TGF-beta oligonucleotide 271tcggagagag atccgtc
1727217DNAArtificial Sequencemodified TGF-beta oligonucleotide
272atcccacgga aataacc 1727317DNAArtificial Sequencemodified
TGF-beta oligonucleotide 273ctcagtatcc cacggaa 1727417DNAArtificial
Sequencemodified TGF-beta oligonucleotide 274actgccgaga gcgcgaa
1727517DNAArtificial Sequencemodified TGF-beta oligonucleotide
275ctgatgtgtt gaagaac 1727617DNAArtificial Sequencemodified
TGF-beta oligonucleotide 276tgaggtatcg ccaggaa 1727717DNAArtificial
Sequencemodified TGF-beta oligonucleotide 277actgccgcac aactccg
1727817DNAArtificial Sequencemodified TGF-beta oligonucleotide
278cggcccacgt agtacac 1727916DNAArtificial Sequencemodified
TGF-beta oligonucleotide 279cccagcggca acggaa 1628016DNAArtificial
Sequencemodified TGF-beta oligonucleotide 280tcgcgccaag aggtcc
1628116DNAArtificial Sequencemodified TGF-beta oligonucleotide
281ggtcggcgac tcccga 1628216DNAArtificial Sequencemodified TGF-beta
oligonucleotide 282gtcggagaga gatccg 1628316DNAArtificial
Sequencemodified TGF-beta oligonucleotide 283tcagtatccc acggaa
1628416DNAArtificial Sequencemodified TGF-beta oligonucleotide
284cgagagcgcg aacagg 1628516DNAArtificial Sequencemodified TGF-beta
oligonucleotide 285actgccgaga gcgcga 1628616DNAArtificial
Sequencemodified TGF-beta oligonucleotide 286ggcgtcagca ccagta
1628716DNAArtificial Sequencemodified TGF-beta oligonucleotide
287ggtttccacc attagc 1628816DNAArtificial Sequencemodified TGF-beta
oligonucleotide 288gaggtatcgc caggaa 1628916DNAArtificial
Sequencemodified TGF-beta oligonucleotide 289aaccactgcc gcacaa
1629016DNAArtificial Sequencemodified TGF-beta oligonucleotide
290cggcccacgt agtaca 1629115DNAArtificial Sequencemodified TGF-beta
oligonucleotide 291cggcggctcg tctca 1529215DNAArtificial
Sequencemodified TGF-beta oligonucleotide 292cccagcggca acgga
1529315DNAArtificial Sequencemodified TGF-beta oligonucleotide
293tcgcgccaag aggtc 1529415DNAArtificial Sequencemodified TGF-beta
oligonucleotide 294cgtcgcgcca agagg 1529515DNAArtificial
Sequencemodified TGF-beta oligonucleotide 295ggagcaagcg tcccc
1529615DNAArtificial Sequencemodified TGF-beta oligonucleotide
296gtgcgcccga ggtct 1529715DNAArtificial Sequencemodified TGF-beta
oligonucleotide 297gtctaggatg cgcgg 1529815DNAArtificial
Sequencemodified TGF-beta oligonucleotide 298cagtatccca cggaa
1529915DNAArtificial Sequencemodified TGF-beta oligonucleotide
299ccgagagcgc gaaca 1530015DNAArtificial Sequencemodified TGF-beta
oligonucleotide 300ggcgtcagca ccagt 1530115DNAArtificial
Sequencemodified TGF-beta oligonucleotide 301gttgctgagg tatcg
1530215DNAArtificial Sequencemodified TGF-beta oligonucleotide
302accactgccg cacaa 1530315DNAArtificial Sequencemodified TGF-beta
oligonucleotide 303cggcccacgt agtac 1530414DNAArtificial
Sequencemodified TGF-beta oligonucleotide 304ctcggcgact cctt
1430514DNAArtificial Sequencemodified TGF-beta oligonucleotide
305agcggcaacg gaaa 1430614DNAArtificial Sequencemodified TGF-beta
oligonucleotide 306tcgcgccaag aggt 1430714DNAArtificial
Sequencemodified TGF-beta oligonucleotide 307tccccggcgg caaa
1430814DNAArtificial Sequencemodified TGF-beta oligonucleotide
308tgcgcccgag gtct 1430914DNAArtificial Sequencemodified TGF-beta
oligonucleotide 309gtctaggatg cgcg 1431014DNAArtificial
Sequencemodified TGF-beta oligonucleotide 310ggtcggagag agat
1431114DNAArtificial Sequencemodified TGF-beta oligonucleotide
311cacggaaata acct 1431214DNAArtificial Sequencemodified TGF-beta
oligonucleotide 312agagcgcgaa cagg 1431314DNAArtificial
Sequencemodified TGF-beta oligonucleotide 313atagtcccgc ggcc
1431414DNAArtificial Sequencemodified TGF-beta oligonucleotide
314tagtagtcgg cctc 1431514DNAArtificial Sequencemodified TGF-beta
oligonucleotide 315atagatttcg ttgt 1431614DNAArtificial
Sequencemodified TGF-beta oligonucleotide 316gaggtatcgc cagg
1431714DNAArtificial Sequencemodified TGF-beta oligonucleotide
317gccgcacaac tccg 1431813DNAArtificial Sequencemodified TGF-beta
oligonucleotide 318tcgcgccaag agg 1331913DNAArtificial
Sequencemodified TGF-beta oligonucleotide 319aagcgtcccc ggc
1332013DNAArtificial Sequencemodified TGF-beta oligonucleotide
320gacgccgtgt agg 1332113DNAArtificial Sequencemodified TGF-beta
oligonucleotide 321gtcggcgact ccc 1332213DNAArtificial
Sequencemodified TGF-beta oligonucleotide 322tgcgcccgag gtc
1332313DNAArtificial Sequencemodified TGF-beta oligonucleotide
323gtcggagaga gat 1332413DNAArtificial Sequencemodified TGF-beta
oligonucleotide 324tcccacggaa ata 1332513DNAArtificial
Sequencemodified TGF-beta oligonucleotide 325tgccgagagc gcg
1332613DNAArtificial Sequencemodified TGF-beta oligonucleotide
326tagtcccgcg gcc 1332713DNAArtificial Sequencemodified TGF-beta
oligonucleotide 327tagtagtcgg cct 1332813DNAArtificial
Sequencemodified TGF-beta oligonucleotide 328catagatttc gtt
1332913DNAArtificial
Sequencemodified TGF-beta oligonucleotide 329tttaacttga gcc
1333013DNAArtificial Sequencemodified TGF-beta oligonucleotide
330gaggtatcgc cag 1333113DNAArtificial Sequencemodified TGF-beta
oligonucleotide 331actccggtga cat 1333213DNAArtificial
Sequencemodified TGF-beta oligonucleotide 332gcccacgtag tac
1333312DNAArtificial Sequencemodified TGF-beta oligonucleotide
333tcggcgactc cc 1233412DNAArtificial Sequencemodified TGF-beta
oligonucleotide 334gtcggcgact cc 1233514DNAArtificial Sequencefake
sequence 335aaaaaaaaaa aaaa 1433616DNAArtificial Sequencefake
sequence 336aaaaaaaaaa aaaaaa 1633717DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 337caggaagcgc tggcaac
1733817DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
338ggtgcatgaa ctcactg 1733917DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 339gtcccctaat ggcttcc
1734017DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
340atctgtcccc taatggc 1734117DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 341ccgggtgctg ttgtaaa
1734217DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
342cctggatcat gtcgaat 1734317DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 343ccctggatca tgtcgaa
1734417DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
344gtagcacctg cttccag 1734516DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 345gggctttcta aatgac 1634616DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 346tgactcccag caggcc
1634716DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
347gtgcatgaac tcactg 1634816DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 348ggtgcatgaa ctcact 1634916DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 349atctgtcccc taatgg
1635016DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
350cgggtgctgt tgtaaa 1635116DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 351ccgggtgctg ttgtaa 1635216DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 352cctggatcat gtcgaa
1635316DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
353ccctggatca tgtcga 1635416DNAArtificial Sequencemodified
TGF-beta3 oligonucleotide 354tttgaatttg atttcc 1635516DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 355gggcctgagc agaagt
1635615DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
356gggggctttc taaat 1535715DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 357tttgtttaca cttcc 1535815DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 358ccagctaaag gtggg
1535915DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
359atggctgggt cccaa 1536015DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 360gagtttttcc ttagg 1536115DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 361aggggtggca aggca
1536215DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
362tgactcccag caggc 1536315DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 363gaagcgctgg caacc 1536415DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 364gtgcatgaac tcact
1536515DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
365gtggtgcaag tggac 1536615DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 366ctaatggctt ccacc 1536715DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 367cccctaatgg cttcc
1536815DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
368atctgtcccc taatg 1536915DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 369gatctgtccc ctaat 1537015DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 370agatctgtcc cctaa
1537115DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
371ggtgctgttg taaag 1537215DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 372ccgggtgctg ttgta 1537315DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 373gatcatgtcg aattt
1537415DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
374cctggatcat gtcga 1537515DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 375ccctggatca tgtcg 1537615DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 376gatttccatc acctc
1537715DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
377ttgaatttga tttcc 1537815DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 378agcagttctc ctcca 1537915DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 379gcctgagcag aagtt
1538015DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
380gggcaagggc ctgag 1538115DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 381cccacacttt cttta 1538215DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 382tagcacctgc ttcca
1538314DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
383cgggggcttt ctaa 1438414DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 384ccattcatgc tttc 1438514DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 385aagcgctggc aacc
1438614DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
386accagagccc tttg 1438714DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 387cccctaatgg cttc 1438814DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 388gtcccctaat ggct
1438913DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
389atctgcccct aat 1339014DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 390agatctgtcc ccta 1439114DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 391cgggtgctgt tgta
1439214DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
392atcatgtcga attt 1439314DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 393ccctggatca tgtc 1439414DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 394cctttgaatt tgat
1439514DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
395ttgcggaagc agta 1439614DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 396gcctgagcag aagt 1439713DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 397gggggctttc taa
1339813DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
398agcgctggca acc 1339913DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 399cccctaatgg ctt 1340013DNAArtificial
Sequencemodified TGF-beta3 oligonucleotide 400tcccctaatg gct
1340113DNAArtificial Sequencemodified TGF-beta3 oligonucleotide
401tcatgtcgaa ttt 1340213DNAArtificial Sequencemodified TGF-beta3
oligonucleotide 402atcatgtcga att 13
* * * * *